Concentration-dependent self-interaction assay

ABSTRACT

Methods for producing high concentration protein formulations having high stability are provided. Assays for selecting proteins and formulation conditions that have high self-repulsive attributes are used as an early step in the manufacturing process. Specifically, a protein concentration-dependent self-interaction nanoparticle spectroscopy method is employed as a protein colloidal interaction assay.

FIELD

The invention relates generally to the selection of therapeutic proteinsfor development and commercialization; and more specifically to theselection of proteins that have a greater likelihood of readilyachieving a high protein concentration with concomitant lower likelihoodof forming aggregates. The invention specifically relates to assessingprotein colloidal interactions and selecting proteins to formulate athigh concentration.

BACKGROUND

Large biological molecules— “biotherapeutics”—are an important class ofdrugs. Monoclonal antibodies for example offer exquisite therapeuticspecificity, long biological half-life, and high safety profiles.

Biotherapeutics given their size and complexity are difficult tomanufacture and formulate. Most biotherapeutics are formulated aslyophilized solids or liquid formulations, and stability of thesemolecules is an important problem. Stability not only impacts shelf lifeof the final product, but also the manufacturing process.

A large molecule by definition has a large molecular mass. Therefore, afew moles of drug encompass a relatively large mass. For example, anantibody has a molecular mass of about 150,000 grams per mole, whereas asmall molecule drug like atorvastatin has a molecular mass of about 560grams per mole. Therefore, to attain an effective dose, a high mass ofprotein drug is required. As more drugs are formulated for subcutaneousinjection, that large mass has to fit into a small volume. Toaccommodate therapeutically effective doses, subcutaneous formulationsof biotherapeutic drugs must attain high (about 50 mg/mL or more) orvery high (about 100-250 mg/mL) concentrations, all while keepingviscosity at a manageable level.

As large molecules are crammed into a small space and the distancebetween protein molecules decreases, the frequency of colloidalinteractions, i.e., the through-space effect of one molecule on another,increases. Protein self-interactions (colloidal interactions) aregenerally energetically weak, typically reversible and non-specific. Theinteractions are protein-dependent and are affected by pH, salt andother additives. In some cases the interactions may be net attractive,repulsive, or neutral. The nature and magnitude of the interaction candepend on the concentration of the protein.

Colloidal interactions span a broad spectrum of interactions (potentialenergy) that are quantified with virial coefficients. B₂₂ representspairwise interactions, B₂₂₂ represents 3-body interactions, B₂₂₂₂represents 4-body interactions, and so on. A positive virial coefficientvalue represents repulsive interactions, a negative virial coefficientrepresents attractive interactions, and a zero virial coefficientrepresents an ideal state.

Colloidal interactions are generally present in protein solutions thatexceed ˜2 mg/mL and become significant in solutions that exceed ˜40mg/mL. At the lower concentrations, steric and electrostatic forces tendto dominate. The situation becomes more complex with increasing proteinconcentration. As the concentration of protein increases duringproduction and formulation, colloidal interactions become problematic.

Colloidal interactions impact a variety of downstream processes duringprotein production. Those forces may be beneficial or detrimentaldepending on nature and magnitude of the interactions. Colloidalinteractions affect chromatographic performance, ultrafiltration anddiafiltration (UF/DF), dialysis, viscosity and solution handling, andstability of the protein in solution. Colloidal interactions also impactlong term stability during storage, where oligomers and multimers, aswell as protein aggregates can form. Assessment of a protein's virialcoefficient therefore provides important information before committingresources to the development of a particular protein therapeutic.

Virial coefficient analysis is an established and highly active area ofinvestigation. Current methods of assessing virial coefficient (B₂₂, orthe closely related A₂ ) for particular proteins include membraneosmometry, sedimentation equilibrium analytical ultra-centrifugation,self-interaction chromatography, static light scattering (SLS),diffusion or sedimentation interaction parameters, and self-interactionnanoparticle spectroscopy (SINS).

For example, Cohen and Benedek (U.S. Pat. No. 4,080,264A, published Mar.3, 1978) describe a quasi-elastic light scattering spectroscopic methodto measure antigen or antibody aggregation. Publicover and Vincze (U.S.Pat. No. 7,514,938B2, published Apr. 7, 2009) describe the use ofdielectric relaxation spectroscopy (DRS) to probe the interaction andaggregation of micron and sub-micron scale particles coated withprotein, including antibodies. Holman et al., (US20070291265A1,published Dec. 20, 2007) describe a bifurcated fiber optic system formeasuring light scattering and concentration signals to measureaggregation of macromolecules. Obrezanova et al. (mAbs, 7(2): 352-363,2015) describe the use of size exclusion high pressure liquidchromatography (SE-HPLC) and an oligomer detection assay, which is anoptical density microtiter plate antibody capture assay, tosystematically measure the aggregation propensity of over 500antibodies. Geoghegan et al. (mAbs, 8(5): 941-950, 2016) describe theuse of hydrophobic interaction chromatography (HIC) retention time,affinity-capture SINS, and dynamic light scattering to measuremonoclonal antibody self-interaction, viscosity and stability. Anoverview of the current methods used to assess colloidal proteininteractions is provided by Geng, et al. (J Pharm Sci., 103(11):3356-3363, 2014).

Self-interaction nanoparticle spectroscopy (SINS) for the assessment ofmonoclonal antibody self-association is described by Sule at al.,(Biophys. J., 101: 1749-1757, 2011). Briefly, antibodies are adsorbedonto gold nanoparticles, which are then combined with a buffer in96-well microtiter plates. As the nanoparticles aggregate due to theself-association of the adherent antibodies, the absorbance spectrum ofthe nanoparticles changes. This change in surface plasmon resonancecorrelates with particle association. The assay provides a binaryreadout of “self-association” or “no self-association” based on whetherthe absorbance peak shifts or changes in amplitude. Sule et al. (Mol.Pharmaceutics, 10: 1322-1331, 2013) describe an improved SINS methodcalled affinity-capture self-interaction nanoparticle spectroscopy(AC-SINS). Here, human polyclonal antibodies are first fixed to thenanoparticles, then the coated particles are contacted with lowconcentration/low purity antibody samples, which are then captured bythe anti-human coating. AC-SINS enables the rapid screening of cellsupernatants without the need for extensive antibody purification.AC-SINS, like SINS, provides a binary assessment of positiveself-association or no self-association of the subject antibodies.

Proteins are complex molecules and often show unpredictable behaviorunder varying conditions. The state-of-the art provides some means forobserving protein self-association, but does not provide a rapid andaccurate means to determine the propensity of a protein to undergoself-association under myriad different conditions. Some proteins arestable under some conditions, but unstable under other conditions. Thereremains a need in the art for an assay to determine the dynamic range ofa protein's attractiveness and repulsiveness under changing conditions.

SUMMARY

The inventors developed a method for determining the potential of aprotein to self-assemble (i.e., the protein's inherent repulsiveness orattractiveness) across a dynamic range of conditions. Consequently themethod may be understood as a method for determining the stability ofself-associated proteins or a method for determining the stability ofaggregated proteins. As is further elaborated herein, the protein may bea single type of protein or a combination of any proteins of differentorigins. The method, called concentration-dependent self-interactionnanoparticle spectroscopy (CD-SINS) measures the propensity of a proteinto self-assemble under changing concentration and various ionic strengthand pH conditions. The method enables the prediction of the protein athigh or very high concentration conditions and informs the selection ofproteins for commercial development. The method also provides forinformation on how to formulate a pharmaceutical composition comprisinghigh concentrations of one or more biopharmaceutical drugs whilstmaintaining long shelf life without association of the biopharmaceuticaldrug or drugs. The method also enables for information on how toformulate a composition fulfilling required specifications on physicalproperties such as e.g. a suitable rheology/viscosity to allow for aspecific desired route of administration. The invention also allows fora more rapid screening of biopharmaceuticals using less amount ofmaterial to arrive at a suitable formulation or selecting suitablecandidates for further drug development.

In a first aspect, the invention provides bioanalytical mixturecomprising a plurality of nanoparticles, a protein, and a buffered salt.The protein exists in at least two phases in the mixture: an adherentphase, and a soluble phase. The adherent phase includes proteins thatare adhered to (i.e., coating) the nanoparticles. The soluble phaseincludes proteins that are dissolved in the buffered salt solution. Insome embodiments, the protein exists in a third phase, where theproteins are self-associated as dimer, trimers, tetramers, or higherorder multimers, including aggregated coated nanoparticles.

In one embodiment, the nanoparticle is a gold nanoparticle. In someembodiments, the nanoparticle has a diameter of about 20 nm to about 100nm. In a particular embodiment, the nanoparticle is a gold nanoparticlehaving a diameter of about 20 nm.

In one embodiment, each nanoparticle is coated with protein. In someembodiments, most or all of the nanoparticle surfaces are saturated withprotein, meaning that the surface is completely occupied by protein andno bare surface remains for a protein to adhere. In some embodiments,most or all of the nanoparticle surfaces are saturated with proteinprior to adding other components to the mixture, such as a suitable saltor a buffered salt.

In one embodiment, the mixture contains about 6×10¹¹ to about 7×10¹¹nanoparticles per milliliter of mixture. In a particular embodiment, themixture contains about 6.3×10¹¹ nanoparticles per mL. While not wishingto be bound by theory and assuming that an average protein can bemodeled as a 10 nm sphere, the theoretical upper limit for the number of10 nm spheres needed to coat a 20 nm sphere is about 30. Approximately15 to 20 antibody molecules can be estimated to bind a single 20 nmnanoparticle. Therefore, —2.5 μg/mL of an antibody is estimated to bethe minimum concentration necessary to completely cover 6.3×10¹¹nanoparticles (20 nm)/mL. In some embodiments, the mixture comprisesabout 2 μg/mL to about 512 μg/mL of protein.

In some embodiments, the protein included in the mixture is atherapeutic antibody. In some embodiments, the protein comprises animmunoglobulin Fc domain. In some embodiments, the protein is anantigen-binding protein. Antigen-binding proteins include antibodies,antibody fragments, antibody derivative, Fc-fusion proteins and receptor—Fc-fusion proteins. In one embodiment, the protein is a monoclonalantibody. In a more specific embodiment, the monoclonal antibody is ahuman or humanized antibody. In some embodiments, the monoclonalantibody may be a monospecific antibody or a bispecific antibody.

In some embodiments, the buffered salt comprises a buffer and a salt. Insome embodiments, the buffer provides ionic strength. In someembodiments, the salt buffers the mixture. The salt may in principle beany suitable salt known in the art and may be e.g. any chloride,bromide, phosphate, sulfate or ammonium salt, or any combinationsthereof. Non-limiting examples are e.g sodium chloride, potassiumchloride, potassium phosphate, ammonium chloride etc. In one embodiment,the salt comprises sodium chloride. The salt (e.g., sodium chloride) maybe present at a concentration of about 2 mM to about 300 mM. Inparticular embodiments, the salt is present in the mixture at aconcentration of about 2 mM, about 20 mM, or about 200 mM.

In one embodiment, the buffer comprises 2-(N-morpholino)ethanesulfonicacid (MES). In a specific embodiment, the MES is present in the mixtureat a concentration of about 10 mM and a pH of about 6.

In a second aspect, the invention provides a method for determining thepotential of a protein to self-associate. In one embodiment, the methodcomprises the steps of combining a protein, a plurality ofnanoparticles, and a buffered salt to form a sample: exciting the samplewith light; measuring the light transmitted through the sample; andcalculating the first absorbance intensity ratio of the sample. Theprocess is repeated at least one more time, each time changing one ormore parameters, and obtaining a second, third, etc. absorbance ratio.The multiple absorbance intensity ratios obtained from a given proteinmay be plotted for analysis. Those parameters include salt type (i.e.,neutral, chaotropic, kosmotropic), salt concentration, pH, proteinconcentration, the inclusion or exclusion of additional ingredients.When the absorbance intensity ratio exceeds a threshold value, theprotein is considered to be favorable for dispensing at highconcentration. A protein considered to be favorable for dispensing athigh concentration is expected to remain stable at a high concentrationand to be less prone to aggregation.

In some embodiments, the protein is a therapeutic antibody. In someembodiments, the protein comprises an immunoglobulin Fc domain. In someembodiments, the protein is an antigen-binding protein. Antigen-bindingproteins include antibodies, antibody fragments, antibody derivative,Fc-fusion proteins and receptor —Fc-fusion proteins. In one embodiment,the protein is a monoclonal antibody. In a more specific embodiment, themonoclonal antibody is a human or humanized antibody. In someembodiments, the monoclonal antibody may be a monospecific antibody or abispecific antibody.

In some embodiments, the protein is added to the sample to a finalconcentration of about 2 μg/mL to about 512 μg/mL.

In some embodiments, the nanoparticle is a gold nanoparticle. In someembodiments, the gold nanoparticle has a diameter of about 20 nm toabout 100 nm. In one embodiment, the diameter of the gold nanoparticleis about 20 nm.

In some embodiments, the nanoparticles are added to the sample to afinal concentration of about 5×10¹¹ to about 8×10¹¹ nanoparticles permL, about 6×10¹¹ to about 6.5×10″ nanoparticles per mL, or about6.3×10¹¹ nanoparticles per mL.

The buffered salt may contain a buffer, a buffer that confers ionicstrength, a salt, a salt that has buffering capacity, or a salt and abuffer. In one embodiment, the salt comprises sodium chloride. The salt(e.g., sodium chloride) may be present at a concentration of about 2 mMto about 300 mM. In some specific embodiments, the salt is present inthe mixture at a concentration of about 2 mM, about 5 mM, about 10 mM,about 20 mM, about 50 mM, about 75 mM, about 100 mM, about 110 mM, about120 mM, about 150 mM, about 175 mM, about 200 mM, or about 300 mM.

In one embodiment, the buffer comprises 2-(N-morpholino)ethanesulfonicacid (MES). In a specific embodiment, the MES is present in the mixtureat a concentration of about 10 mM and a pH of about 6.

In some embodiments, the excitation light is white light comprisingwavelengths spanning the visible spectrum. In some embodiments, thetransmitted light is measured at multiple wavelengths ranging from about450 nm to about 750 nm.

The absorbance intensity ratio is a measure of the relative intensity oflight absorbance relative to a standard or control. The control may byexternal or internal. In one embodiment, the absorbance intensity ratiois calculated by dividing the maximum intensity (optical density orabsorbance) of the peak absorbance wavelength of the sample by theinitial absorbance intensity observed. In one embodiment, the initialobserved absorbance intensity is the absorbance intensity observed at450 nm. In that embodiment, the absorbance intensity ratio is peakabsorbance (A_(peak)/absorbance at 450 nm (A₄₅₀ A_(init)). In thatembodiment, the threshold value of the absorbance intensity ratio isabout 1.5 to about 2. In a specific embodiment, the threshold value ofthe absorbance intensity ratio is about 1.7.

In one embodiment, a protein considered favorable for dispensing at highconcentration is combined with an excipient to form a formulated drugsubstance (FDS) or drug product (DP). In one embodiment, the protein isformulated to a final concentration of about 50 mg/mL to about 250mg/mL.

In one embodiment the excipient includes a tonicifier, a buffer, asurfactant, a stabilizer, or any combination of two or more thereof. Inone embodiment, the tonicifier is a salt. In a specific embodiment, thesalt is sodium chloride. In one embodiment, the buffer buffers at aboutpH 6 to about pH 7. In a specific embodiment, the buffer is histidine.In another specific embodiment, the buffer is phosphate. In oneembodiment, the surfactant is a polysorbate, such as polysorbate 20 orpolysorbate 80. In one embodiment, the stabilizer is a sugar, such assucrose or trehalose. In another embodiment, the stabilizer is an aminoacid, such as proline or arginine.

In a third aspect, the invention provides a method of manufacturing atherapeutic protein. In one embodiment, the method comprises the step ofselecting a protein having high colloidal stability from a plurality ofdifferent proteins of varying unknown colloidal stability; producing theselected protein in a host cell; purifying the protein; and combiningthe protein at a high concentration with an excipient to form aformulated drug substance or drug product where the protein is stable.In one embodiment, less than 10% of the protein in the formulated drugsubstance or drug product is aggregated.

In one embodiment, the step of selecting the protein having highcolloidal stability comprises the steps of combining the protein with ananoparticle and a buffered salt to form a sample; exciting the samplewith light; measuring the light transmitted by the sample; andcalculating the absorbance intensity ratio of the sample. This selectingstep is repeated one or more times with one or more parameters beingchanged. The parameters that are changed include salt type, saltconcentration, pH, protein concentration, and the inclusion or exclusionof additional ingredients. When the absorbance intensity ratio exceeds athreshold value the protein is selected as having high colloidalstability.

In some embodiments, the excitation light is white light comprisingwavelengths spanning the visible spectrum. In some embodiments, thetransmitted light is measured at multiple wavelengths ranging from about450 nm to about 750 nm.

The absorbance intensity ratio is a measure of the relative intensity oflight absorbance relative to a standard or control. The control may byexternal or internal. In some embodiments, the absorbance intensityratio is calculated by dividing the maximum intensity (optical densityor absorbance) of the peak absorbance wavelength of the sample by (1)the initial absorbance intensity observed in that sample (internal), orby (2) the initial absorbance intensity observed in a sample ofnanoparticles the absence of protein (external). In one embodiment, theinitial observed absorbance intensity is the absorbance intensityobserved at 450 nm. In that embodiment, the absorbance intensity ratiois peak absorbance (A_(peak))/absorbance at 450 nm (A₄₅₀ or A_(init)).In that embodiment, the threshold value of the absorbance intensityratio is about 1.5 to about 2. In a specific embodiment, the thresholdvalue of the absorbance intensity ratio is about 1.7.

In a further embodiment, the invention relates to a compositioncomprising a biotherapeutic drug (such as e.g. a protein or antibody) ina high concentration. Specifically, the composition may be such that nomore than about 10% of the total protein species is present as anirreversible aggregate at the concentration of the biotherapeutic drug.Alternatively, the composition is such that the threshold value asdiscussed herein is e.g. in range of e.g. about 1.5 to about 2.0(A_(peak)/A₄₅₀). A further alternative is where the threshold value ise.g. about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0(A_(peak)/A_(control)). In some embodiments, the threshold value is inrange of about 0.7 to about 1.0 (A_(peak)/A_(control)). A furtheralternative is where the threshold value is e.g. about 0.7, about 0.8,about 0.9, or about 1.0 (A_(peak)/A_(control)). In yet a furtherembodiment, the composition is such that no more than about 10% of thetotal protein species is present as an irreversible aggregate at theconcentration of the biotherapeutic drug and such that the compositionhas a threshold value as discussed herein is e.g. in range of e.g. about1.5 to about 2.0 (A_(peak)/A₄₅₀) or where the threshold value is e.g.about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0(A_(peak)/A₄₅₀), and/or where the threshold value is in range of about0.7 to about 1.0 (A_(peak)/A_(control)), or where the threshold value ise.g. about 0.7, about 0.8, about 0.9, or about 1.0(A_(peak)/A_(control)). The concentration of the biopharmaceutical drugmay be about 50 mg/mL or more. Alternatively, the concentration of thebiopharmaceutical drug may be in range of e.g. about 50 mg/mL to about500 mg/mL, such as e.g. about 50 mg/mL to about 250 mg/mL, such as e.g.about 100 mg/mL to about 250 mg/mL.

In one embodiment the invention relates to a composition obtainable bythe method for determining the potential of a protein to self-assembleas disclosed herein.

In yet a further aspect, the invention relates to a compositioncomprising a biotherapeutic drug (such as e.g. a protein or antibody) ina high concentration for use in medicine. Depending on the drug, aperson skilled in the art will know which or what clinical conditionsthat may be treated by administering the composition to a subject inneed thereof. The composition may be be such that no more than about 10%of the total protein species is present as an irreversible aggregate atthe concentration of the biotherapeutic drug. Alternatively, thecomposition is such that the threshold value as discussed herein is e.g.in range of e.g. about 1.5 to about 2.0 (A_(peak)/A₄₅₀). A furtheralternative is where the threshold value is e.g. about 1.5, about 1.6,about 1.7, about 1.8, about 1.9, or about 2.0 (A_(peak)/A₄₅₀). A In someembodiments, the threshold value is in range of about 0.7 to about 1.0(A_(peak)/A_(control)). A further alternative is where the thresholdvalue is e.g. about 0.7, about 0.8, about 0.9, or about 1.0(A_(peak)/A_(control)). In yet a further embodiment, the composition issuch that no more than about 10% of the total protein species is presentas an irreversible aggregate at the concentration of the biotherapeuticdrug and such that the composition has a threshold value as discussedherein is e.g. in range of e.g. about 1.5 to about 2.0 (A_(peak)/A₄₅₀)or where the threshold value is e.g. about 1.5, about 1.6, about 1.7,about 1.8, about 1.9, or about 2.0 (A_(peak)/A₄₅₀), and/or where thethreshold value is in range of about 0.7 to about 1.0(A_(peak)/A_(control)), or where the threshold value is e.g. about 0.7,about 0.8, about 0.9, or about 1.0 (A_(peak)/A_(control)). Theconcentration of the biopharmaceutical drug may be about 50 mg/mL ormore. Alternatively, the concentration of the biopharmaceutical drug maybe in range of e.g. about 50 mg/mL to about 500 mg/mL, such as e.g.about 50 mg/mL to about 250 mg/mL, such as e.g. about 100 mg/mL to about250 mg/mL. The composition may be administered as a single dose ormultiple doses as considered needed for obtaining desired result intreatment. Alternatively, the invention relates to a compositioncomprising a biotherapeutic drug (such as e.g. a protein or antibody) ina high concentration for manufacture of a medicament in the treatment ofa disease or diseases treatable or curable by said biotherapeutic drug.

In one embodiment, the invention provides a method of preparing abiopharmaceutical composition (such as e.g. a protein or an antibody)such that the resulting composition possesses a suitable rheology.Specifically, the method for determining the potential of a protein toself-assemble as disclosed herein allows for determination of an arrayof provision of a stable composition comprising a high biopharmaceuticaldrug concentration. One important attribute of such composition is theviscosity. Consequently, the method of the invention will allow for thepreparation of a composition with desired physical properties such thatthe composition may be administered via a desired route or mode ofadministration. One exemplary administration route may be administrationby injection. In such instance, it is thus important to have a viscosityof the prepared composition enabling injection by e.g. a syringe andcannula. Such cannula may be a cannula of size such as e.g. 6G, 8G, 9G,10G, 11G 12G, 13G, 14G, 16G, 19G, 20G, 21G, 22G, 23G, 24G, or 26G. Theterm “viscosity” refers to the dynamic or absolute viscosity (at 20° C.and normal pressure) which is a measure of the resistance of a fluidwhich is being deformed by either shear stress or extensional stress.“Viscosity” thus describes a fluid's internal resistance to flow and maybe thought of as a measure of fluid friction. Consequently, the lessviscous something is, the greater its ease of movement (fluidity). Inthe present context, the relevant ranges of viscosity are from about10-10.000 mPa·s, such as e.g. about 20-9000 mPa·s, such as e.g. about30-8000 mPa·s, such as e.g. about 40-7000 mPa·s, such as e.g. about50-6000 mPa·s, such as e.g. about 70-5000 mPa·s, such as e.g. about90-4000 mPa·s, such as e.g. about 100-3000 mPa·s or about 10 mPa·s, orabout 20 mPa·s, or about 30mPa·s, or about 40 mPa·s, or about 50mPa·s oralternatively from about 1 mPa·s to about 20 mPa·s, such as about 2mPa·s, such as about 3 mPa·s, such as about 4 mPa·s, such as about 5mPa·s such as about 6 mPa·s, such as about 7 mPa·s such as about 10mPa·s, about 13 mPa·s, about 15 mP·s, such as e.g. about 20 mPa·s.

Other units for measurement of viscosity are well-known, for example, 1centipoise is equivalent to 1 mPa·s. Without being bound to anyparticular theory, a typical protein at a low concentration (i.e. lessthan or equal to 10 mg/mL) exhibits a viscosity of about 10 cP. As such,a highly concentrated protein composition, e.g. an antibody composition,exhibiting a viscosity below about 10 centipoise (below about 10 mPa·s)is highly suitable for use as a biopharmaceutical composition. A proteincomposition (at a high concentration) having a viscosity from about 10to about 15 centipoise (about 10 mPa·s to about 15 mPa·s) is safe toproceed with in the manufacturing and drug development process. Aprotein composition (at a high concentration) having a viscosity fromabout 15 to about 20 centipoise (about 15 mPa·s to about 20 mPa·s)informs to proceed with caution in the manufacturing and drugdevelopment process. A protein composition (at a high concentration)having a viscosity greater than about 20 centipoise (greater than about20 mPa·s) informs of problematic manufacturing and drug developmentprocessing. The viscosity may be manipulated by the various componentsand ingredients as disclosed herein. “Soluble” and “highly soluble” alsorefer to a protein having a suitable viscosity for any of the usesdisclosed herein, e.g. for making and using a biopharmaceuticalcomposition.

In one embodiment, the protein is an antigen-binding protein, forexample an antibody, an antibody fragment or a receptor-Fc-fusionprotein.

In one embodiment, the host cell in which the protein is made is aChinese hamster ovary (CHO) cell or a derivative of a CHO cell, such asa CHO-K1 cell or an EESYR®, cell (see Chen et al., US771997B2, publishedAug. 10, 2010).

In one embodiment, the step of purifying the protein comprisessubjecting the protein to (1) one or more step of affinitychromatography, ion exchange chromatography, hydrophobic interactionchromatography, mixed mode chromatography, and hydroxyapatitechromatography; and (2) one of both of ultrafiltration anddiafiltration.

In one embodiment, the protein is formulated at a final concentrationthat is at least 50 mg/mL. In one embodiment, the concentration of theprotein is about 50 mg/mL to about 250 mg/mL.

In one embodiment the excipient with which the protein is combined toform the formulated drug substance or drug product comprises one or moreof a tonicifier, a buffer, a surfactant, and stabilizer. In oneembodiment, the tonicifier is a salt. In a specific embodiment, the saltis sodium chloride. In one embodiment, the buffer buffers at about pH 6to about pH 7. In a specific embodiment, the buffer is histidine. Inanother specific embodiment, the buffer is phosphate. In one embodiment,the surfactant is a polysorbate, such as polysorbate 20 or polysorbate80. In one embodiment, the stabilizer is a sugar, such as sucrose ortrehalose. In another embodiment, the stabilizer is an amino acid, suchas proline or arginine.

DRAWINGS

FIG. 1 depicts an absorbance profile of dispersed 20 nm goldnanoparticles (line A) and agglomerated 20 nm gold nanoparticles (lineB). The Y- axis depicts optical density in arbitrary absorbance units.The X-axis depicts transmitted light wavelength in nanometers (nm).

FIG. 2 depicts a scatter plot of absorbance intensity ratios ofdifferent concentrations of monoclonal antibody 2 (mAb2) and 20 nm goldnanoparticles in 2 mM salt (circles), 20 mM salt (squares) and 200 mMsalt (triangles). The Y-axis depicts the ratio of peak absorbanceintensity of each sample condition over the initial absorbanceintensity. The X-axis depicts the concentration of mAb2 in microgramsper milliliter.

FIG. 3 depicts a scatter plot of absorbance intensity ratios ofdifferent concentrations of monoclonal antibody (mAb) 1 (open symbols)and mAb5 (closed symbols) in the presence of 20 nm gold nanoparticles in2 mM salt (squares), 20 mM salt (triangles) and 200 mM salt (circles).The Y-axis depicts the ratio of peak absorbance intensity of each samplecondition over the initial absorbance intensity. The X-axis depicts theconcentration of mAb1 in micrograms per milliliter. The boxed area tothe left (A) represents conditions of higher colloidal repulsiveconditions having high selection value, the boxed area in the middle (B)represents conditions of mixed repulsive and attractive conditionshaving cautious or mixed selection value, and the boxed area to theright (C) represents conditions of attractiveness having problematic orlow selection value.

FIG. 4 depicts an overlay of multiple absorbance profiles of dispersed20 nm gold nanoparticles combined with human serum albumin (HSA) ofvarying concentration (3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50μg/mL, 100 μg/mL. 200 μg/mL and 400 μg/mL in order from top to bottom atright-most [750 nm] portion of curve). The Y- axis depicts arbitraryabsorbance in arbitrary units. The X-axis depicts transmitted lightwavelength in nanometers (nm).

FIG. 5 depicts a scatter plot of absorbance intensity ratios ofdifferent concentrations of HSA derived from the raw absorbance spectradepicted in FIGS. 4 and 20 nm gold nanoparticles in 2 mM salt (circles),20 mM salt (squares) and 200 mM salt (triangles). The Y-axis depicts theratio of peak absorbance intensity of each sample condition over theinitial absorbance intensity. The X-axis depicts the concentration ofHSA in micrograms per milliliter.

FIG. 6 depicts a scatter plot of absorbance intensity ratios ofdifferent concentrations of monoclonal antibody 1 (mAb1) and 20 nm goldnanoparticles in 2 mM salt (circles), 20 mM salt (squares) and 200 mMsalt (triangles). The Y-axis depicts the ratio of peak absorbanceintensity of each sample condition over the initial absorbanceintensity. The X-axis depicts the concentration of mAb1 in microgramsper milliliter.

FIG. 7 depicts a scatter plot of scattered light intensity of solutionsof mAb1 at different concentrations and ionic salt concentrations: 2 mMNaCl (circles), 20 mM NaCl (squares), and 200 mM NaCl (triangles). TheY-axis represents light scattering intensity in arbitrary absorbanceunits. The X-axis represents concentration of mAb1 in grams per liter.The solid line represents an ideal hard sphere of comparable diameter.

FIG. 8 depicts a scatter plot of absorbance intensity ratios ofdifferent concentrations of monoclonal antibody 6 (mAb6) and 20 nm goldnanoparticles in 2 mM salt (circles), 20 mM salt (squares) and 200 mMsalt (triangles). The Y-axis depicts the ratio of peak absorbanceintensity of each sample condition over the initial absorbanceintensity. The X-axis depicts the concentration of mAb6 in microgramsper milliliter.

FIG. 9 depicts a scatter plot of scattered light intensity of solutionsof mAb6 at different concentrations and ionic salt concentrations: 2 mMNaCl (circles), 20 mM NaCl (squares), and 200 mM NaCl (triangles). TheY-axis represents light scattering intensity in arbitrary absorbanceunits. The X-axis represents concentration of mAb6 in grams per liter.The solid line represents an ideal hard sphere of comparable diameter.

FIG. 10 depicts a scatter plot of absorbance intensity ratios ofdifferent concentrations of monoclonal antibody 3 (mAb3) and 20 nm goldnanoparticles in 2 mM salt (circles), 20 mM salt (squares) and 200 mMsalt (triangles). The Y-axis depicts the ratio of peak absorbanceintensity of each sample condition over the initial absorbanceintensity. The X-axis depicts the concentration of mAb3 in microgramsper milliliter.

FIG. 11 depicts a scatter plot of absorbance intensity ratios ofdifferent concentrations of monoclonal antibody 4 (mAb4) and 20 nm goldnanoparticles in 2 mM salt (circles), 20 mM salt (squares) and 200 mMsalt (triangles). The Y-axis depicts the ratio of peak absorbanceintensity of each sample condition over the initial absorbanceintensity. The X-axis depicts the concentration of mAb4 in microgramsper milliliter.

FIG. 12 depicts a scatter plot of absorbance intensity ratios ofdifferent concentrations of monoclonal antibody 5 (mAb5) and 20 nm goldnanoparticles in 2 mM salt (circles), 20 mM salt (squares) and 200 mMsalt (triangles). The Y-axis depicts the ratio of peak absorbanceintensity of each sample condition over the initial absorbanceintensity. The X-axis depicts the concentration of mAb5 in microgramsper milliliter.

FIG. 13 depicts a CD-SINS scatter plot of absorbance intensity ratios ofdifferent concentrations of monoclonal antibody 7 (mAb7) formulated indifferent buffers, pHs, and ionic strengths. Each symbol represents adifferent buffer, pH and ionic strength formulation with variable mAb7concentrations (X-axis).

FIG. 14 depicts a CD-SINS scatter plot of absorbance intensity ratios ofdifferent concentrations of monoclonal antibody 7 (mAb7) formulated inthe presence of different sugars, concentrations of sugars, and/or aminoacids. Each symbol represents a different sugar/amino acid formulationwith variable mAb7 concentrations (X-axis).

FIG. 15 depicts a CD-SINS scatter plot of absorbance intensity ratios ofdifferent concentrations of monoclonal antibody 7 (mAb7) (8 μg/mL, 32μg/mL, 128 μg/mL, and 512 μg/mL mAbK) combined with different benzoatecompounds. When combined with ρ-aminobenzoic acid [PABA (-∘-), opencircle], mAb7 exhibited a favorable dynamic colloidal interactionprofile in which the absorbance intensity ration (Y-axis) exceeds 1.6.

FIG. 16 depicts a CD-SINS scatter plot of absorbance intensity ratios ofdifferent concentrations of monoclonal antibody K (mAbK) in the presenceof varying concentrations of ρ-aminobenzoic acid (PABA). The X-axisdepicts log 2 of the concentration of mAb7 in micrograms per milliliter.The Y-axis depicts the absorbance intensity ratio. Open circles (-∘-)represent no PABA, open squares (-□-) represent 12 mM PABA, opentriangles (-Δ-) represent 18 mM PABA, closed circles (-•-) represent 24mM PABA, closed squares (-▪-) represent 30 mM PABA, and closed triangles(- ▴-) represent 36 mM PABA.

FIG. 17 depicts a dot plot of the steady shear viscosity (in mPa*s) of amAb7 solution containing no PABA (closed circles └-•-┘) or 20 mM PABA(closed triangles └-▴-┘) as a function of mAb7 concentration in g/L.Open symbols represent extrapolated viscosity of solutions expected tocontain 100 g/L mAb7.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about”, when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than15%. For example, as used herein, the expression “about 100” includes 85and 115 and all integer and non-integer values in between (e.g, 86,86.001, 87, 88, 88.3, 89, etc. . . . ).

Absolute amounts and relative amounts of excipients, ingredients, andother materials may be described by mass, or moles. Units of mass may beexpressed as grams, milligrams, micrograms, and the like). The term“weight” as in “weight/volume” or “w/v” means “mass”. Relative amountsmay be expressed as percent weight (i.e., percent mass), wherein one (1)percent weight to volume (w/v) means 1 gram of material per 100milliliter of volume. Also for example, one (1) part ingredient “A” perone (1) part ingredient “B” by weight means e.g. that for every one (1)gram of ingredient “A” there is one (1) gram of ingredient “B”. Also forexample, one percent (1%) by weight of ingredient “A” means e.g. thatfor every 100 grams of total mass of a particle there is one (1) gram ofingredient “A”. Relative amounts of an ingredient may also be expressedin terms of moles or number of molecules per given volume, e.g.,millimoles per liter (millimolar (mM)), or per other ingredient, e.g., Xpart ingredient “A” per Y part ingredient “B” by mole means for every Xmoles of “A” there are Y moles of “B”

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the exemplar methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference intheir entirety.

Assay to Determine Dynamic Colloidal Stability: Concentration-DependentSelf-Interaction Nanoparticle Spectroscopy (CD-SINS)

An improvised SINS-based assay for determining the dynamic virial rangefor proteins is provided. A protein exhibits complex behavior, which isvery sensitive to the chemical physical environment of the protein.Therefore, assessing the virial coefficient of a protein under aparticular set of environmental circumstances is insufficient todetermine the virial coefficient of that protein in other environments.Obtaining multiple virials for a given protein under myriad conditionsis painstaking and time consuming. The inventors herein disclose a highthrough put assay to determine the dynamic colloidal stability of aprotein, which informs the selection of a protein that can be formulatedor otherwise kept at a high concentration without having problemsassociated with aggregation, or at least minimizing potentialaggregation problems.

That assay employs SINS in a novel way by assessing nanoparticleaggregation under conditions of varying protein concentration, varyingionic strength, varying pH, and other conditions. As described above andin Sules (2011), gold nanoparticles are coated with protein and placedinto buffered solution. As the nanoparticles aggregate due to proteinself-association, the surface plasmon resonance of the particleschanges: the maximum absorbance shifts to the higher wavelength (redshift); and the absorbance is of a lower intensity, i.e., the absorbanceprofile spreads out and moves left (FIG. 1). Here, the uncoated 20 nmgold nanoparticles have a peak absorbance at about 520 to 530 nm.

A comparison of conventional SINS with the well-established static lightscattering (SLS) method of determining B₂₂ or A₂ shows general agreementfor most antibodies that were tested. Table 1 compares the peakabsorbance of beads coated with specific different monoclonal antibodiesand human serum albumin (HSA) under the SINS assay to the A₂ virials forthe same proteins determined by SLS. As shown in Table 1, SINS analysisreadily distinguishes attractive (negative A₂) from repulsive (positiveA₂) systems. However, the conventional SINS and SLS assays show nodynamic range for repulsive conditions. Also, the few discrepanciesbetween conventional SINS and SLS indicate complex behavior of someproteins.

TABLE 1 Nature of 2 mM NaCl 20 mM NaCl 200 mM NaCl Protein system SINSA₂ SINS A₂ SINS A₂ HSA Repulsive 526  1.17E−4 526  9.68E−5 528  7.85E−5*mAb1 Repulsive 530  1.72E−4 531  6.70E−5 532  1.34E−5  mAb2 Mixed 583−2.36E−5 536 −1.44E−5 534  1.47E−5  mAb3 Attractive 556 −3.39E−5 555−4.68E−5 565 NP mAb4 Repulsive 531 −5.90E−5 533 −1.06E−5 535  1.38E−5 mAb5 Mixed 562 −1.09E−5 535 −2.14E−5 530 −7.67E−6 

The inventors observed that some proteins are capable of being stable athigh concentrations, but show negative A₂ or red-shifted maximumabsorbance spectra (λ_(max)). for example, mAb5 can readily attain aconcentration of about ˜200 g/L and remain stable, yet its A₂ isnegative and its tines is red-shifted at the lower ionic strength.Similarly, mAb2 can readily attain a concentration of about ˜175 g/L andremain stable, yet its A₂ is negative at lower salt concentration andits λ_(max) is red-shifted at the lower ionic strength.

Here, an improved method of SINS is disclosed. The method providesdynamic colloidal stability data for proteins in order to assessprotein-specific phenomena earlier in the development timeline, whilecircumventing the need for large quantities of protein. The methodemploys coated nanoparticle surface plasmon resonance in the presence ofvarying amounts of protein in solution in excess of the amounts requiredto coat the nanoparticles. Absorbance profiles are observed, absorbanceintensity ratios calculated for each variant sample, and plotted todetermine the dynamic colloidal stability determined.

In one embodiment, for each sample in a plurality of samples used todetermine multiple individual absorbance intensity ratio values for agiven subject protein, nanoparticles are combined with varying amountsof protein in excess of the minimum amount of protein necessary tocompletely coat the particles. The amount of minimum protein useddepends upon the molecular mass of the protein (i.e., its hydrodynamicradius), the size (surface area) of the nanoparticle, and theconcentration of nanoparticles. For example, when 20 nm goldnanoparticles are used at about 6 to 6.5×10″ particles per mL, about 2.5μg/mL of a protein of about 50 to 150 kDa is sufficient to fully coatthe nanoparticles. Therefore, protein in excess of 2.5 μg/mL is used foreach sample of the plurality of samples under those conditions. Here,for example protein in included in a sample at about 2.6 μg/mL- about512 μg/mL or more, about 3 ±1 μg/mL, about 4±1 μg/mL, about 5±1 μg/mL,about 6 ±1 μg/mL, about 7±1 μg/mL, about 8±1 μg/mL, about 9±1 μg/mL,about 10±1 μg/mL, about 15±5 μg/mL, about 20±5 μg/mL, about 25±5 μg/mL,about 30±5 μg/mL, about 40±5 μg/mL, about 50±10 μg/mL, about 60±10μg/mL, about 70±10 μg/mL, about 80±10 μg/mL, about 90±10 μg/mL, about100±10 μg/mL, about 125±15 μg/mL, about 150±25 μg/mL, 175±25 μg/mL,200±25 μg/mL, 225±25 μg/mL, 250±25 μg/mL, 300±50 μg/mL, 350±50 μg/mL,400±50 μg/mL, 450±50 μg/mL, 500±50 μg/mL or 512±50 μg/mL. In someembodiments, the plurality of samples includes two samples, threesamples, four samples, five samples, six samples, seven samples, eightsamples, nine samples, 10 samples or more containing a different proteinconcentration.

For example, the plurality of samples in one embodiment includes6.3×10¹¹ particles/mL 20 nm gold nanoparticles and an antibody at afirst concentration of about 3.125 μg/mL, a second concentration of 6.25μg/mL, a third concentration of about 12.5 μg/mL, a fourth concentrationof about 25 μg/mL, a fifth concentration of about 50 μg/mL, a sixthconcentration of about 100 μg/mL, a seventh concentration of about 200μg/mL, and an eighth concentration of about 400 μg/mL.

In one embodiment, for each sample in the plurality of samples used todetermine multiple individual absorbance intensity ratio values for agiven subject protein, each nanoparticle/protein combination is combinedwith a variable salt concentration. For example, the sample may containa neutral salt protein, like sodium chloride, at a concentration ofabout 1 about 10 μM, about 100 μM, about 1 mM, about 2 mM, about 4 mM,about 6 mM, about 8 mM, about 10 mM, about 20 mM, about 30 mM, about 40mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM,about 100 mM, about 150 mM, about 175 mM, about 200 mM, about 250 mM orabout 300 mM or more. In another embodiment, the salt may be achaotropic salt, such as guanidinium chloride, lithium perchlorate,lithium aceteate, magnesium chloride or the like. The sample may alsocontain in addition to or in lieu of the chaotropic salt anotherchaotropic agent such as butanol, ethanol, phenol, propanol, sodiumdodecyl sulfate, thiourea or urea. In another embodiment, the sample maycontain a kosmotropic salt such as a salt of carbonate, sulfate orphosphate, for example ammonium sulfate.

The plurality of samples may contain two, three, four, five, six, seven,eight, nine or 10 or more subsets of samples having a different saltconcentration. Thus for example, a plurality of samples may comprise 10different protein concentrations at three different salt concentrationsfor a total of 30 samples. In a specific embodiment, one subset of theplurality of samples contains about 2 mM sodium chloride, another set ofthe plurality of samples contains about 20 mM sodium chloride, andanother set of plurality of samples contains about 200 mM sodiumchloride. 2 mM salt is considered low ionic strength and 200 mM isconsidered high ionic strength. High tonicity up to and including 300 mMsalt, although not pertinent to most therapeutic formulations ofprotein, is used in the context of protein preparations undergoingpurification via hydrophobic interaction chromatography (HIC) columns.Therefore the invention is useful in determining whether the highconcentration protein in high concentration salt conditions will besuitable for HIC purification or the like.

The white light is applied to the each sample, and its absorbancespectrum is obtained. The absorbance intensity ratio is calculated fromeach sample of the plurality and plotted. In those samples in which thenanoparticles do not aggregate or wherein the aggregation is low, i.e.,where the protein exhibits repulsiveness or low aggregation potential,the absorbance peak wavelength and absorbance intensity are similar tothe control uncoated nanoparticles. In those samples in which thenanoparticles aggregate, i.e., where the protein exhibits attractivenessor high aggregation potential, the absorbance peak wavelength shiftstoward the red and the absorbance profile flattens as the peakabsorbance intensity decreases relative to the control uncoatednanoparticles. Each sample of the plurality of samples for a givenprotein may exhibit different absorbance profiles (i.e., differentabsorbance intensity ratios), in some cases showing attractiveness andin other cases showing repulsiveness. Those proteins have a dynamicrange of colloidal stability and may be colloidally stable underspecific conditions. For some proteins, the protein may showrepulsiveness across all parameters (samples). Such a protein isconsidered robust or having robust dynamic colloidal stability. Forother proteins, the protein may show attractiveness under all testedparameter conditions. Such a protein is considered colloidally instable.

Those proteins that exhibit repulsiveness under at least one testedcondition (in at least one sample) may be selected for large scaleproduction and formulation as a stable drug substance. Those proteinsare less likely to form aggregates during production and storage.Furthermore, those conditions under which a protein exhibitsrepulsiveness can inform the production steps and formulation excipientsof the protein as a drug substance. For example, utilizingconcentration-dependent SINS (CD-SINS) as described herein can determinemanufacturability of proteins, e.g. in the downstream ultrafiltrationand diafiltration steps. Selection of a low viscosity protein solutionfor large scale manufacturing may increase efficiency and decrease costin bioprocessing (Shire SJ, 2009, “Formulation and manufacturability ofbiologics.” Curr Opin Biotechnol. 20(6):708-14). CD-SINS is alsogenerally amenable to a wide range of excipients (both traditional andnon-traditional). CD-SINS can be utilized to derive structure-activityrelationships between different chemical series used used forformulating the protein drug substance, and can be utilized to selectfor excipients that reverse self-association (see Example 11 and FIGS.13 and 14).

General criteria for selecting molecules and conditions havingacceptable colloidal stability dynamics are depicted in FIG. 3, whichplots the absorbance intensity ratio of two proteins, mAb1 and mAb5under three ionic strength conditions. Three buckets are provided: (A)safe to proceed, which includes robust molecules expected to be stableat high concentration; (B) proceed with caution, which includesmolecules showing absorbance intensity ratios above the threshold level(e.g., an A_(peak)/A₄₅₀ of ≥1.7) under some conditions; and (C)potentially problematic, which includes molecules failing to attain thethreshold absorbance intensity ratio value under any condition. Forexample, mAb1 (open symbols) is robust in terms of colloidal stability,falling into the “safe to proceed” bucket under lower salt conditions (2mM NaCl, open squares; 20 mM NaCl, open triangles), a “proceed withcaution” category under higher salt conditions (200 mM NaCl, opencircles). Conversely, mAb5 (closed symbols) is less robust, falling intothe “potentially problematic” category under lower salt conditions, andthe “proceed with caution” bucket under the higher salt condition.

Concentration-dependent SINS (CD-SINS) demonstrates how protein/solventsystems “evolve” with increasing protein concentration. It capturesmyriad colloidal interactions that different proteins exhibit at highprotein concentration: repulsive to ideal (neutral); attractive toideal; ideal to attractive; and insensitive. It captures many of thedifferent facets of colloidal interactions such as charge mediatedrepulsion or attraction, tracts electrostatic screening andqualitatively provides a fairly wide dynamic range. CD-SINS revealspossible hydrophobic mediated issues and flags generally problematicmolecules. The method provides an analytical tool for assessing highprotein concentration developability with remarkably minimal proteinrequirements, and which is amenable to automation.

Reduction of Viscosity of Attractive or Mixed Colloidal Protein

Those proteins having unfavorable dynamic colloidal interactionprofiles, i.e., that tend to self-associate, generally may have highviscosities at higher concentrations. Such high viscosity at highconcentration may render the protein undesirable for parenteralinjection. The CD-SINS assay described herein is useful to screen forviscosity-reducing excipients. In one aspect, a method for producing areduced-viscosity protein formulation is provided. In one embodiment, apotential viscosity-reducing excipient

In some embodiments, the viscosity-reducing excipient reduces theviscosity of the protein solution by at least 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,2.5-fold, 3- fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, or >5-foldrelative to the viscosity of the protein solution without theviscosity-reducing excipient.

In some embodiments, the viscosity-reducing excipient is an amino acidor a salt of an amino acid. In some embodiments, the viscosity-reducingexcipient is a hydrocarbon, an alkane, an alkene, and alkyne, a fattyacid, a fatty acid tail, a benzene-containing structure, a benzoic acid,a substituted hydrocarbon, a substituted alkane, a substituted alkene, asubstituted alkyne, a substituted fatty acid, a substituted fatty acidtail, a substituted benzene-containing structure, a substituted benzoicacid, a sulfonic acid, an aminobenzoic acid, an alkylated benzoic acid,an hydroxybenzoic acid, or an ammonium salt of a benzoic acid. In oneembodiment, the viscosity-reducing excipient is a para-amino benzoicacid (PABA). In one embodiment, the PABA is included in the proteinformulation at a concentration of 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM,11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21mM, 22 mM, 23 mM, 24 mM, or 25 mM. In one embodiment, theviscosity-reducing excipient is PABA combined at a concentration of >12mM or at a concentration of 20 mM in the protein solution.

Definitions

As used herein, the term “colloidal stability”, which can be usedinterchangeably with “propensity to self-associate” or “propensity toaggregate”, means the net (overall) effect of through-space molecularforces, for example electrostatic, van der Waals, and the like, whichmay result in an attractive, neutral, or repulsive interaction potentialof a protein.

As used herein, the term “absorbance intensity ratio” means the ratio ofthe peak absorbance intensity of a sample to baseline absorbanceintensity. The baseline intensity may be obtained from the absorbance ofthe sample at an arbitrary wavelength sufficiently shorter or longerthan the expected absorbance wavelength. In some embodiments, theexpected absorbance wavelength for the 20 nm gold nanoparticles rangesfrom about 500 to 600 nm, and peaks at about 530 nm. Therefore, thebaseline absorbance intensity may be the absorbance intensity of thesample at a wavelength lower than 500 or greater than 600. For example,the baseline absorbance intensity may be the absorbance intensity of thesample at 450 nm. Here, the absorbance intensity ratio is the absorbanceintensity at the peak absorbance wavelength (A_(peak)) of the sampledivided by the absorbance intensity at 450 nm (A₄₅₀ or Ainitial).

In other embodiments, the baseline absorbance intensity may be theintensity of the peak absorbance of uncoated nanoparticles (controlnanoparticles). Here, the nanoparticles without protein, but in the samebuffered salt in which the experimental coated nanoparticles aresampled, are subjected to absorbance analysis. The peak absorbanceintensity of the uncoated control nanoparticles (A_(control)) serves asthe baseline absorbance intensity. Here, the absorbance intensity ratiois calculated as A_(peak)/A_(control).

Absorbance is generally expressed in arbitrary units, which cancel outwhen the ratio is calculated. Absorbance is measured by passing a whitelight source through a sample. The sample absorbs light of certainwavelengths at certain strengths, according to the optical properties ofthe sample. The intensity of light that passes through the sample, i.e.,the “transmitted light”, is measured at multiple wavelengths and theintensity of the “absorbed light” is calculated.

The absorbance spectrum of the gold nanoparticles is determined by theparticle surface plasmon resonance. The electric field from incominglight interacts with the free electrons on the surface of gold particlesand results in a strong absorption in the visible region. This opticalproperty depends on the size, shape, and agglomeration status of thenanoparticles. For example, smaller particles (20 nm in diameter) absorbat a lower wavelength (522 nm) and with a narrower and more intense peak(A=˜0.6) than larger particles (e.g., 250 nm particle absorbs moststrongly at 570- 660 nm with an intensity about 40% of the intensity ofthe 20 nm particle). An irregularly-shaped particle shows a broader peakof lower intensity that is red-shifted relative to a spherical particle.Likewise, agglomerated particles show a broader peak of lower intensitythat is red-shifted relative to discrete dispersed spherical particles(FIG. 1).

As used herein, the term “threshold value” denotes an absorbanceintensity ratio above which a protein is considered to be feasible forachieving a stable high concentration in solution. A protein samplehaving an absorbance ratio below the threshold value indicates that theprotein, at least under the experimental conditions of pH, ionicstrength, and protein density, may not be feasible for achieving astable high concentration in solution. This feasibility of a protein toachieve a high concentration in solution is inversely related to theprotein's “attractiveness” or potential for self-association, and ispositively related to the protein's “repulsiveness”. In someembodiments, the threshold value is about 1.5, 1.6, 1.7, 1.8, 1.9, or 2(A_(peak)/A₄₅₀). In some embodiments, the threshold value is about 0.7,0.8, 0.9, or 1 (A_(peak)/A_(control)).

As used herein, the term “dynamic range” refers to a protein's potentialfor self-association over a range of conditions. Prior artself-association assays that employ self-interaction nanoparticlespectroscopy (SINS) only measure the self-association potential ofproteins under a single set of conditions. A protein is deemed“attractive” under that single point assay may be rejected, whereas aprotein that is deemed “repulsive” under that assay may be retained asfeasible for high concentration formulation and production. Here, aseries of absorbance intensity ratios are obtained under varyingexperimental conditions and plotted, generating a “dynamic range”profile for a particular protein. Under some conditions, the protein mayhave a below-threshold absorbance intensity ratio, while under otherconditions the protein may have an above-threshold absorbance intensityratio. Such a protein would have an acceptable degree of repulsivenessunder those conditions, which can inform the selection of proteinmanufacturing processes and formulation excipients and concentrations.For example, monoclonal antibody 2 (mAb2) shows an acceptable degree ofrepulsiveness under high concentration/high ionic strength (e.g., 200 mMNaCl) conditions, but unacceptable attractiveness under low ionicstrength conditions (FIG. 2).

Experimental conditions used to determine the dynamic range of theprotein include the concentration of the protein; the size, shape numberof nanoparticle; the material from which the nanoparticle is made; themethod of coating the nanoparticles with protein; the nature and amountsof ingredients in the buffer or solvent; the pH; and the ionic strengthof the solution. For example, the pH of the buffer may affect theoverall charge of the protein depending on the protein's isoelectricpoint (pI), and the charge in turn may affect the second order virialcoefficient of the protein (negative B₂₂ is attractive, and a positiveB₂₂ is repulsive). Tonic strength (i.e., salt content) is generallyknown to affect protein self-attraction, where increasing ionic strengthshields proteins against charge-based repulsion and promotes attraction.

In some embodiments, data points (i.e., absorbance intensity ratios) aretaken at different salt concentrations or with no salt at all. Forexample, the salt, e.g., sodium chloride, may be included at anyconcentration ranging from trace amounts to supersaturated. In someembodiments, sodium chloride is included in the “buffered salt” at aconcentration of about 0.1 mM to about 1 M, 1 mM to 500 mM, or 2 mM to300 mM. In some embodiments, data points are taken at two, three, four,five, six, seven, eight, nine, 10 or more different salt concentrationconditions. In one embodiment, at least three salt concentrationconditions are used, for example 2 mM, 20 mM and 200 mM sodium chloride.In another embodiment, at least five salt concentration conditions areused. Non-limiting examples of five salt concentration conditions mayinclude: 1 mM, 2 mM, 20 mM, 200 nM and 300 mM sodium chloride, or 1 mM,5 mM, 20 mM, 100 nM and 300 mM sodium chloride. The skilled artisan mayreadily adapt the assay by using different salts and/or salt conditionsbased on the nature of the experimental or therapeutic use of theprotein of interest.

As used herein, the term “buffered salt” denotes an aqueous solutioncontaining a buffer and a salt. The salt may be at any concentration,and the buffer may have buffering capacity at any range of pH. In someembodiments, the salt and the buffer may be one and the same, such ascalcium carbonate. Salts are known to affect protein interaction and areoften used to precipitate proteins, to affect protein folding, tostabilize pharmaceutical formulations, to provide tonicity, and toregulate protein behavior during chromatography. Salts may bekosmotropic (positive free energy of hydrogen bonding) or chaotropic(negative free energy of hydrogen bonding) agents. Kosmotropesfacilitate water-water interactions and are commonly used to salt-outproteins. Examples of kosmotropic ions include carbonate, sulfate,phosphate, magnesium, lithium, zinc and aluminum. Chaotropes disrupthydrogen bonding and weaken hydrophobic effects, thereby promotingprotein denaturation. Examples of chaotropic salts include guanidiniumchloride, lithium perchlorate, lithium acetate and magnesium chloride.Salts like sodium chloride are near the middle of the Hofmeister series,meaning that they are neither effective at salting-in nor salting-out.Depending on the objective of the protein self-association assay, saltsof the buffered salt may be selected for their ability to salt-in,salt-out or remain effectively neutral at physiological concentrations.

“Buffers” are included to control pH, which in turn affects the chargeproperties of proteins, and subsequent structure and function. Dependingon the objective of the protein self-association assay, buffers may beselected for example for their ability to enhance protein purification,promote long term protein stability or allow for patient comfort duringadministration of a therapeutic protein. Useful buffers are well-knownin the art and include MES, TRIS, PIPES, MOPS, phosphate,citrate-phosphate, citrate, acetate, carbonate-bicarbonate, histidine,imidazole and the like. In one particular embodiment, the buffer used inthe protein self-association assay is MES(2-(N-morpholino)ethanesulfonic acid), which has a useful buffer rangeof about pH 5.5 to 6.7. In a specific embodiment, the MES is included inthe buffered salt at about 10 mM, pH6.

A buffer is used in the buffered salt component of the assay system andreagents. A buffer is also used to control the pH of solutions thatcontain the protein beyond the assay. Buffers are used in cell cultureproduction media used to produce the protein. Buffers are also used insolutions used during protein purification and the various unitoperations deployed therein. Buffers are used in formulated drugsubstances and in the final drug product formulation.

Buffers useful in the formulation of proteins are well known in the artand include histidine, succinate, citrate, acetate, phosphate and thelike. The buffer may be included in the formulated drug substance (FDS)or drug product (DP) at a concentration of from 1 mM to 100 mM. In someparticular embodiments, the buffer is included at about 10 mM. Incertain embodiments, the buffer is present at a concentration of 5mM±0.75 mM to 15 mM±2.25 mM; 6 mM ±0.9 mM to 14 mM ±2.1 mM; 7 mM ±1.05mM to 13 mM ±1.95 mM; 8 mM ±1.2 mM to 12 mM ±1.8 mM, 9 mM ±1.35 mM to 11mM 1.65 mM; 10 mM f 1.5 mM; or about 10 mM. In some specificembodiments, the buffer system of the FDS or DP comprises histidine,phosphate, and/or acetate at 10 mM ±1.5 mM.

In some embodiments, the buffer is selected from a chemical capable ofbuffering somewhere within the pH range of about 3 to about 9, or withinthe pH range of about 3.7 to about 8.0. For example, the pre-lyophilizedsolution may have a pH of about 3.4, about 3.6, about 3.8, about 4.0,about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, about 5.2, about5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6,about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, orabout 8.0.

The buffer may be a combination of individual buffers, such as, e.g.,the combination of histidine and acetate (his-acetate buffer). In oneembodiment, the buffer has a buffering range of about 3.5 to about 6, orabout 3.7 to about 5.6, such as the range buffered by acetate. In oneembodiment, the buffer has a buffering range of about 5.5 to about 8.5,or about 5.8 to about 8.0, such as the range buffered by phosphate. Inone embodiment, the buffer has a buffering range of about 5.0 to about8.0, or about 5.5 to about 7.4, such as the range buffered by histidine.

As used herein, the term “nanoparticle” refers to a spherical, nearspherical or spheroidal particle having a diameter on the scale of 10⁻⁹M(0.001 micron) to 10⁻⁶ M (1 micron). Nanoparticles may comprise anymaterial, including organic polymers, metals, semiconductor materials,magnetic materials and combinations of materials. Metal nanoparticlesare particularly suitable for surface plasmon resonance-based assays,since metal spheres have free electrons on their surface that caninteract with the electric field from incident light, resulting in astrong absorbance spectrum. Gold nanoparticles and silver nanoparticlesare examples of metal nanoparticles useful for measuring changes inplasmon resonance. The size, shape and material of the nanoparticleaffects the intensity and wavelength of maximum absorbance. Goldnanoparticles of 20 nm to 400 nm diameter are useful. In someembodiments, the nanoparticle used in the self-association dynamic rangeassay is a gold nanoparticle (AuNP) with a diameter of about 20 nm, 30nm, 40 nm, 50 nm, 60 nm, 80 nm or 100 nm.

As used herein, the term “light” means electromagnetic radiation (EMR).The light can be of a single wavelength, of a narrow range ofwavelengths, of a broad range of wavelengths such as “white light”, of acollection of wavelengths. The light can be in the form of a laser beamor as diffused light. The self-aggregation assay is a spectrophotometricassay, where the affect that the sample has on light is measured. Thus,light is applied to the sample (i.e., “incident light”), the sampleinteracts with the light, such as generating plasmons and absorbingparticular wavelengths of light (i.e., absorbed light), and a subset ofthe light is transmitted through the sample and on to a detector (i.e.,“transmitted light”). In some embodiments, the incident light is a whitelight comprising EMR having wavelengths of 400 nm to 800 nm. In someembodiments, transmitted light is detected and measured at wavelengthsspanning 400 nm to 800 nm, from which the absorbance intensity isdetermined.

As used herein, the term “self-associate” refers to the non-specificbinding of a specific protein to another protein of the same species. Bynon-specific, what is meant is association by weak forces that areconsidered to by non-biological. To distinguish non-specific fromspecific interaction, the association of two identical antibody halvesto form an intact antibody is considered a specific interaction.Conversely, the associations of two or more identical intact antibodiesvia Van der Waals or hydrophobic interactions to form dimers, trimers orhigher order multimers that are reversible or irreversible are“non-specific”.

As used herein, the term “stable” or “stability” refers to the retentionof an acceptable degree of physical structure (thermodynamic stability),chemical structure (kinetic stability), or biological function(functional stability) of a protein or other biological macromoleculeover time. The protein may be stable even though it does not maintain100% of its physical structure, chemical structure, or biologicalfunction after storage for a certain amount of time. Under certaincircumstances, maintenance of about 90%, about 95%, about 96%, about97%, about 98% or about 99% of the protein's structure or function afterstorage for a particular amount of time may be regarded as “stable”.

Stability may be measured by determining the percentage of nativeprotein remaining in a sample. The percentage of protein that retainsits native form may be determined by size exclusion chromatography,which separates high molecular weight aggregates of the protein form thelower molecular weight native protein. A stable protein will retain 90%or more of its native structure over time. A stable protein contains nomore than 10% of the total protein species as an irreversible aggregate.

A stable protein has a low rate of aggregate formation. A stable proteinundergoes an increase in the formation of high molecular weight species,i.e., aggregation, that is less than 15%, less than 14%, less than 13%,less than 12%, less than 11%, less than 10%, less than 9%, less than 8%,less than 7%, less than 6%, less than 5%, less than 4%, less than 3%,less than 2%, less than 1%, or less than 0.5% during storage at about 5°C. to about 25° C. for up to 7 months, up to 8 months, up to 9 months,up to 10 months, up to 11 months, up to 12 months, up to 13 months, upto 14 months, up to 15 months, up to 16 months, up to 17 months, up to18 months, up to 19 months, up to 20 months, up to 21 months, up to 22months, up to 23 months, or up to 24 months.

Other methods may be used to assess the stability of a protein such as,e.g., differential scanning calorimetry (DSC) to determine thermalstability, controlled agitation to determine mechanical stability, andabsorbance at about 350 nm or about 405 nm to determine solutionturbidities. In one embodiment, a protein may be considered stable ifafter storage for 6 months or more at about 5° C. to about 25° C., thechange in OD405 of the formulation is less than about 0.05 (e.g., 0.04,0.03, 0.02, 0.01, or less) from the OD405 of the protein at time zero.

As used herein, the term “protein” means any amino acid polymer havingmore than about 50 amino acids covalently linked via amide bonds.Proteins contain one or more amino acid polymer chains known in the artas “polypeptides”. A protein may contain one or more polypeptides toform a single functioning biomolecule. “Polypeptides” generally containover 50 amino acids, whereas “peptides” generally contain 50 amino acidsor less. Proteins may contain one or more covalent and non-covalentmodifications. Disulfide bridges (i.e., between cysteine residues toform cystine) may be present in some proteins. These covalent links maybe within a single polypeptide chain, or between two individualpolypeptide chains. For example, disulfide bonds are essential to properstructure and function of insulin, immunoglobulins, protamine, and thelike. For a recent review of disulfide bond formation, see Oka andBulleid, “Forming disulfides in the endoplasmic reticulum,” 1833(11)Biochim Biophys Acta 2425-9 (2013).

In addition to disulfide bond formation, proteins may be subject toother post-translational modifications. Those modifications includelipidation (e.g., myristoylation, palmitoylation, farnesoylation,geranylgeranylation, and glycosylphosphatidylinositol (GPI) anchorformation), alkylation (e.g., methylation), acylation, amidation,glycosylation (e.g., addition of glycosyl groups at arginine,asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and/ortryptophan), and phosphorylation (i.e., the addition of a phosphategroup to serine, threonine, tyrosine, and/or histidine). For a recentreview on the post-translational modification of proteins produced ineukaryotes, see Mowen and David, “Unconventional post-translationalmodifications in immunological signaling,” 15(6) Nat Immunol 512-20(2014); and Blixt and Westerlind, “Arraying the post-translationalglycoproteome (PTG),” 18 Curr Opin Chem Biol. 62-9 (2014).

Examples of proteins include therapeutic proteins, recombinant proteinsused in research or therapy, trap proteins and other receptor Fc-fusionproteins, chimeric proteins, antibodies, monoclonal antibodies, humanantibodies, bispecific antibodies, antibody fragments, nanobodies,recombinant antibody chimeras, cytokines, chemokines, peptide hormones,and the like. Proteins may be produced using recombinant cell-basedproduction systems, such as the insect bacculovirus system, yeastsystems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHOderivatives like CHO-K1 cells). For a recent review discussingtherapeutic proteins and their production, see Ghaderi et al.,“Production platforms for biotherapeutic glycoproteins. Occurrence,impact, and challenges of non-human sialylation,” 28 Biotechnol GenetEng Rev. 147-75 (2012).

As used herein, the term “antigen-binding protein” denotes any proteinthat binds another molecular entity. The molecular entity may be apeptide, polypeptide, protein, epitope, hapten, antigen or biologicalmolecule. For example, an antigen-binding protein includes a receptormolecule that binds a ligand, where the ligand is the antigen.Antigen-binding proteins include antibodies, antibody fragments (e.g.,Fabs), single-chain antibodies, ScFv molecules, recombinant proteinscomprising receptors and parts of receptors, ligand molecules,recombinant proteins comprising ligands or parts of ligands, recombinantmolecules comprising multiple receptors or receptor fragments (e.g.,receptor-Fc-fusion proteins), and the like.

“Antibody” or “immunoglobulin molecule” is a subset or subtype of anantigen-binding protein. The canonical immunoglobulin protein (e.g.,IgG) comprises four polypeptide chains, two heavy (H) chains and twolight (L) chains inter-connected by disulfide bonds. Each heavy chaincomprises a heavy chain variable region (abbreviated herein as HCVR orVH) and a heavy chain constant region. The heavy chain constant regioncomprises three domains, CH1, CH2 and CH3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region comprisesone domain, CL. The VH and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRsmay be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may beabbreviated as LCDR1, LCDR2 and LCDR3. As used herein, “monoclonalantibody” merely means an antibody of single molecular entity, usuallyproduced by clones of a parent cell that produces a single antibody.Monoclonal antibodies are distinguished from polyclonal antibodies bythe fact that polyclonal antibodies represent a collection of antibodiesmade by different cells. A monoclonal antibody may have monovalentaffinity, meaning that both antibody halves are identical and bind tothe same epitope, or bivalent affinity, as in a “bispecific antibody”,meaning that one antibody half binds a different epitope than the otherantibody half.

“Bispecific antibody” includes any antibody capable of selectivelybinding two or more epitopes. Bispecific antibodies generally comprisetwo different heavy chains, with each heavy chain specifically binding adifferent epitope, either on two different molecules (e.g., antigens) oron the same molecule (e.g., on the same antigen). If a bispecificantibody is capable of selectively binding two different epitopes (afirst epitope and a second epitope), the affinity of the first heavychain for the first epitope will generally be at least one to two orthree or four orders of magnitude lower than the affinity of the firstheavy chain for the second epitope, and vice versa. The epitopesrecognized by the bispecific antibody can be on the same or a differenttarget (e.g., on the same or a different protein). Bispecific antibodiescan be made, for example, by combining heavy chains that recognizedifferent epitopes of the same antigen. For example, nucleic acidsequences encoding heavy chain variable sequences that recognizedifferent epitopes of the same antigen can be fused to nucleic acidsequences encoding different heavy chain constant regions, and suchsequences can be expressed in a cell that expresses an immunoglobulinlight chain. A typical bispecific antibody has two heavy chains eachhaving three heavy chain CDRs, followed by (N-terminal to C-terminal) aCHI domain, a hinge, a CH2 domain, and a CH3 domain, and animmunoglobulin light chain that either does not confer antigen-bindingspecificity but that can associate with each heavy chain, or that canassociate with each heavy chain and that can bind one or more of theepitopes bound by the heavy chain antigen-binding regions, or that canassociate with each heavy chain and enable binding or one or both of theheavy chains to one or both epitopes.

The phrase “heavy chain,” or “immunoglobulin heavy chain” includes animmunoglobulin heavy chain constant region sequence from any organism,and unless otherwise specified includes a heavy chain variable domain.Heavy chain variable domains include three heavy chain CDRs and four FRregions, unless otherwise specified. Fragments of heavy chains includeCDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has,following the variable domain (from N-terminal to C-terminal), a CH1domain, a hinge, a CH2 domain, and a CH3 domain. A functional fragmentof a heavy chain includes a fragment that is capable of specificallyrecognizing an antigen (e.g., recognizing the antigen with a KD in themicromolar, nanomolar, or picomolar range), that is capable ofexpressing and secreting from a cell, and that comprises at least oneCDR.

The phrase “light chain” includes an immunoglobulin light chain constantregion sequence from any organism, and unless otherwise specifiedincludes human kappa and lambda light chains. Light chain variable (VL)domains typically include three light chain CDRs and four framework (FR)regions, unless otherwise specified. Generally, a full-length lightchain includes, from amino terminus to carboxyl terminus, a VL domainthat includes FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4, and a light chainconstant domain. Light chains that can be used with this inventioninclude those, e.g., that do not selectively bind either the first orsecond antigen selectively bound by the antigen-binding protein.Suitable light chains include those that can be identified by screeningfor the most commonly employed light chains in existing antibodylibraries (wet libraries or in silico), where the light chains do notsubstantially interfere with the affinity and/or selectivity of theantigen-binding domains of the antigen-binding proteins. Suitable lightchains include those that can bind one or both epitopes that are boundby the antigen-binding regions of the antigen-binding protein.

The phrase “variable domain” includes an amino acid sequence of animmunoglobulin light or heavy chain (modified as desired) that comprisesthe following amino acid regions, in sequence from N-terminal toC-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. A “variable domain” includes an amino acid sequence capableof folding into a canonical domain (VH or VL) having a dual beta sheetstructure wherein the beta sheets are connected by a disulfide bondbetween a residue of a first beta sheet and a second beta sheet.

The phrase “complementarity determining region,” or the term “CDR,”includes an amino acid sequence encoded by a nucleic acid sequence of anorganism's immunoglobulin genes that normally (i.e., in a wild-typeanimal) appears between two framework regions in a variable region of alight or a heavy chain of an immunoglobulin molecule (e.g., an antibodyor a T cell receptor). A CDR can be encoded by, for example, a germlinesequence or a rearranged or unrearranged sequence, and, for example, bya naive or a mature B cell or a T cell. In some circumstances (e.g., fora CDR3), CDRs can be encoded by two or more sequences (e.g., germlinesequences) that are not contiguous (e.g., in an unrearranged nucleicacid sequence) but are contiguous in a B cell nucleic acid sequence,e.g., as the result of splicing or connecting the sequences (e.g., V-D-Jrecombination to form a heavy chain CDR3).

Fc-containing proteins include antibodies, bispecific antibodies,immunoadhesins, “receptor-Fc-fusion proteins” and other binding proteinsthat comprise at least a functional portion of an immunoglobulin CH2 andCH3 region. A “functional portion” refers to a CH2 and CH3 region thatcan bind an Fc receptor (e.g., an FcγR; or an FcRn, i.e., a neonatal Fcreceptor), and/or that can participate in the activation of complement.If the CH2 and CH3 region contains deletions, substitutions, and/orinsertions or other modifications that render it unable to bind any Fcreceptor and also unable to activate complement, the CH2 and CH3 regionis not functional.

Fc-containing proteins can comprise modifications in immunoglobulindomains, including where the modifications affect one or more effectorfunction of the binding protein (e.g., modifications that affect FcγRbinding, FcRn binding and thus half-life, and/or CDC activity). Suchmodifications include, but are not limited to, the followingmodifications and combinations thereof, with reference to EU numberingof an immunoglobulin constant region: 238, 239, 248, 249, 250, 252, 254,255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307,308, 309, 311, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375,376, 378, 380, 382, 383, 384, 386, 388, 389, 398, 414, 416, 419, 428,430, 433, 434, 435, 437, 438, and 439.

For example, and not by way of limitation, the binding protein is anFc-containing protein and exhibits enhanced serum half-life (as comparedwith the same Fc-containing protein without the recited modification(s))and have a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at 428 and/or 433 (e.g.,L/R/SUP/Q or K) and/or 434 (e.g., H/F or Y); or a modification at 250and/or 428; or a modification at 307 or 308 (e.g., 308F, V308F), and434. In another example, the modification can comprise a 428L (e.g.,M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g, V259I),and a 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434(e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and256E) modification; a 250Q and 428L modification (e.g., T250Q andM428L); a 307 and/or 308 modification (e.g., 308F or 308P).

Some recombinant Fc-containing proteins contain receptors or receptorfragments, ligands or ligand fragments that have cognate bindingpartners in biological systems. “Receptor Fc-fusion proteins” refer torecombinant molecules that contain a soluble receptor fused to animmunoglobulin Fc domain. Some receptor Fc-fusion proteins may containligand binding domains of multiple different receptors. Those receptorFc-fusion proteins are known as “traps” or “trap molecules”. Rilonoceptand aflibercept are examples of marketed traps that antagonize IL1R (seeU.S. Pat. No. 7,927,583) and VEGF (see U.S. Pat. No. 7,087,411),respectively. Other recombinant Fc-containing proteins include thoserecombinant proteins containing a peptide fused to an Fc domain, forexample Centocor's MIMETIBODYTM technology. Recombinant Fc-containingproteins are described in C. Huang, “Receptor-Fc fusion therapeutics,traps, and MTMETIBODY technology,” 20(6) Curr. Opin. Biotechnol. 692-9(2009).

As used herein, the term “tonicifier” or “tonicifying agent” is asubstance or combination of substances that provides tonicity orosmolality to a formulation or solution. The formulation may require aphysiological osmolarity, which is approximately 0.29 osmoles of soluteper kilogram of solvent (290mOsm). A formulation having a physiologicalosmolality is generally referred to as physiologically isotonic. Aformulation may have an osmolality lower than 290 mOsm (physiologicallyhypotonic) or higher than 290 mOsm (physiologically hypertonic). Atonicifier is added to a formulation to adjust the formulation to theappropriate tonicity. The term “tonicity” may be used interchangeablywith osmolality or osmolarity.

Tonicifiers include salts, which are added to adjust ionic strength orconductance, and non-salt tonicifiers. Commonly used salts includesodium chloride, potassium chloride, magnesium chloride, and calciumchloride. Non-salt tonicifiers include sugars, sugar alcohols,monosaccharides, and disaccharides, examples of which include sorbitol,mannitol, sucrose, trehalose, glycerol, maltose, and lactose.

As used herein, the term “surfactant” denotes an additive or excipientthat reduces interfacial surface tension. Some surfactants have alipophilic portion and a hydrophilic portion. Surfactants are believedto provide additional stability by reducing protein-protein hydrophobicinteraction and the resulting formation of high molecular weight species(i.e., aggregates). One or more surfactants may be included inprotein-containing solutions, including FDSs, DPs, protein processingand manufacturing solutions, and protein self-association assaysolutions. Surfactants may be ionic or non-ionic. Non-ionic surfactantsinclude, e.g., alkyl poly(ethylene oxide), alkyl polyglucosides (e.g.,octyl glucoside and decyl maltoside), fatty alcohols such as cetylalcohol and oleyl alcohol, cocamide MEA, cocamide DEA, and cocamide TEA.Specific non-ionic surfactants include, e.g., polyoxyethylene sorbitanesters (a.k.a. polysorbates) such as polysorbate 20, polysorbate 28,polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80,polysorbate 81, and polysorbate 85; poloxamers such as poloxamer 188,poloxamer 407; polyethylene-polypropylene glycol; or polyethylene glycol(PEG). Polysorbate 20 is also known as TWEEN 20, sorbitan monolaurateand polyoxyethylenesorbitan monolaurate. Polysorbate 80 is also known asTWEEN 80, sorbitan monooleate and polyoxyethylenesorbitan monooleate.

The amount of surfactant contained within a protein-containing solutionmay vary depending on the specific properties and purposes desired ofthe solution. In certain embodiments, the solution may contain about0.001% (w/v) to about 0.5% (w/v) surfactant (e.g., polysorbate 20 orpolysorbate 80). For example, solution may contain about 0.001%; about0.0015%; about 0.002%; about 0.0025%; about 0.003%; about 0.0035%; about0.004%; about 0.0045%; about 0.005%; about 0.0055%; about 0.006%; about0.0065%; about 0.007%; about 0.0075%; about 0.008%; about 0.0085%; about0.009%; about 0.0095%; about 0.01%; about 0.015%; about 0.016%; about0.017%; about 0.018%; about 0.019%; about 0.02%; about 0.021%; about0.022%; about 0.023%; about 0.024%; about 0.025%; about 0.026%; about0.027%; about 0.028%; about 0.029%; about 0.03%; about 0.031%; about0.032%; about 0.033%; about 0.034%; about 0.035%; about 0.036%; about0.037%; about 0.038%; about 0.039%; about 0.04%; about 0.041%; about0.042%; about 0.043%; about 0.044%; about 0.045%; about 0.046%; about0.047%; about 0.048%; about 0.049%; about 0.05%; about 0.051%; about0.052%; about 0.053%; about 0.054%; about 0.055%; about 0.056%; about0.057%; about 0.058%; about 0.059%; about 0.06%; about 0.061%; about0.062%; about 0.063%; about 0.064%; about 0.065%; about 0.066%; about0.067%; about 0.068%; about 0.069%; about 0.07%; about 0.071%; about0.072%; about 0.073%; about 0.074%; about 0.075%; about 0.076%; about0.077%; about 0.078%; about 0.079%; about 0.08%; about 0.081%; about0.082%; about 0.083%; about 0.084%; about 0.085%; about 0.086%; about0.087%; about 0.088%; about 0.089%; about 0.09%; about 0.091%; about0.092%; about 0.093%; about 0.094%; about 0.095%; about 0.096%; about0.097%; about 0.098%; about 0.099%; about 0.10%; about 0.15%; about0.20%; about 0.25%; about 0.30%; about 0.35%; about 0.40%; about 0.45%;or about 0.50% surfactant (e.g., polysorbate 20 or polysorbate 80).

As used herein, the term “stabilizer” denotes a molecule or compound, ora combination of chemical entities (i.e., more than one chemical entity)that serves to stabilize the native conformation of the protein.Stabilizers stabilize proteins in solution through one or more of thefollowing mechanisms: (1) increasing the surface tension of water, (2)protein-excipient exclusion which forms a layer of water around theprotein, (3) negative peptide bond interaction, and (4) repulsiveinteraction with the surface of the protein. Regardless of the specificmechanism, stabilizers are preferentially excluded from the proteinsurface, thereby enriching water at the protein surface. Also, theunfavorable protein-excipient interaction renders protein unfoldingthermodynamically unfavorable, since the surface area of the proteinincreases during denaturation. Stabilizers include e.g., polyols,sugars, amino acids, salting out salts, or any combination thereof.Examples of useful stabilizers include polyethylene glycol, sorbitol,glycerol, mannitol, trehalose, sucrose, arginine, alanine, proline,glycine, sodium chloride, or any combination thereof. Sucrose andtrehalose are the most frequently used sugars.

The present invention is further described by the following non-limitingitems.

Item 1. A Method for Determining the Potential of a Protein toSelf-Associate, the Method Comprising:

-   -   a. combining a protein, a nanoparticle, and a buffered salt to        form a sample;    -   b. exciting the sample with light;    -   c. measuring the light transmitted through the sample;    -   d. calculating the absorbance intensity ratio of the sample,

wherein the protein is stable at high concentration when the absorbanceintensity ratio exceeds a threshold value.

Item 2. The Method of Item 1, Wherein the Protein is an Antigen-BindingProtein.

Item 3. The method of item 2, wherein the antigen-binding protein is anantibody, an antibody fragment, or a receptor-Fc-fusion protein.

Item 4. The method of item 3, wherein the antigen-binding protein is ahuman monoclonal antibody.

Item 5. The method according to any one of the preceding items, whereinthe protein is in the sample at a concentration of about 2 μg/mL toabout 512 μg/mL.

Item 6. The method according to any one of the preceding items, whereinthe nanoparticle is a gold nanoparticle.

Item 7. The method of item 6, wherein the gold nanoparticle has adiameter of about 20 nm to about 100 nm.

Item 8. The method of item 7, wherein the diameter of the goldnanoparticle is about 20 nm.

Item 9. The method according to any one of the preceding items, whereinthe sample comprises about 5×10¹¹ to about 8×10¹¹ nanoparticles per mL.

Item 10. The method according to any one of the preceding items, whereinsample comprises about 6-6.5×10¹¹ nanoparticles per mL.

Item 11. The method according to any one of the preceding items, whereinthe salt is present in the sample at a concentration of about 2 mM toabout 250 mM.

Item 12. The method according to any one of the preceding items, whereinthe salt is sodium chloride.

Item 13. The method according to any one of the preceding items, whereinthe salt concentration is about 2 mM, about 20 mM, or about 200 mM.

Item 14. The method according to any one of the preceding items, whereinthe transmitted light is measured at multiple wavelengths ranging fromabout 450 nm to about 750 nm.

Item 15. The method according to any one of the preceding items, whereinthe absorbance intensity ratio is the ratio of the absorbance maximum tothe initial absorbance.

Item 16. The method according to any one of the preceding items, whereinthe threshold value of the absorbance intensity ratio is about 1.7.

Item 17. The Method According to any One of the Preceding Items FurtherComprising Repeating Steps (a)-(d) Using a Different Concentration ofProtein in the Sample.

Item 18. The method according to any one of the preceding items furthercomprising repeating steps (a)-(d) using a different concentration ofsalt in the sample.

Item 19. The method according to any one of the preceding items furthercomprising repeating steps (a)-(d) using a different pH of the sample.

Item 20. The Method According to any One of the Preceding Items FurtherComprising:

e. combining the highly soluble protein at a high concentration with anexcipient to form a formulated drug substance.

Item 21. The method according to any one of the preceding items, whereinthe concentration of the protein is about 50 mg/mL to about 500 mg/mL.

Item 22. The method of according to any of items 20 or 21, wherein theexcipient is selected from the group consisting of a tonicifier, abuffer, a surfactant, stabilizer, and a combination thereof.

Item 23. The method of item 22, wherein the tonicifier is a salt.

Item 24. The method according to any one of the preceding items, whereinthe salt is NaCl.

Item 25.A composition comprising a biotherapeutic drug obtainable by themethod according to any of the preceding claims.

Item 26. The composition according to item 25, wherein no more thanabout 10% of the total biotherapeutic drug species is present as anirreversible aggregate at the concentration of the biotherapeutic drug.

Item 27. The composition according to any of items 25-26, wherein thecomposition is such that the threshold value is in range of e.g. about1.5 to about 2.0 (A_(peak)/A₄₅₀).

Item 28. The composition according to any of items 25-27, wherein thethreshold value is in range of about 0.7 to about 1.0(A_(peak)/A_(control)).

Item 29. The composition according to any of items 25-28, wherein theconcentration of the biopharmaceutical drug is in range of e.g. about 50mg/mL to about 500 mg/mL, about 50 mg/mL to about 250 mg/mL, or about100 mg/mL to about 250 mg/mL.

Item 30. A composition obtainable according to any of items 25-29 foruse in medicine.

Item 31. A method according to any of items 1-24 for preparing acomposition comprising a biopharmaceutical drug, wherein the compositionhas a viscosity from about 10-10.000 mPa·s, about 20-9000 mPa·s, about30-8000 mPa·s, about 40-7000 mPa·s, about 50-6000 mPa·s, about 70-5000mPa·s, about 90-4000 mPa·s, about 100-3000 mPa·s or about 10 mPa·s, orabout 20 mPa·s, or about 30mPa·s, or about 40 mPa·s, or about 50mPa·s oralternatively from about 1 mPa·s to about 20 mPa·s, about 2 mPa·s, about3 mPa·s, about 4 mPa·s, about 5 mPa·s, about 6 mPa·s, about 7 mPa·s,about 10 mPa·s, 13 mPa·s, 15 mP, or about 20 mPa·s.

Item 32. The method according to item 31, wherein the biopharmaceuticaldrug is one or more proteins or one or more antibodies or any mixturesthereof.

Item 33. The method according to any of items 31-32, wherein theantibody is a monoclonal antibody, or a polyclonal antibody, or acombination thereof.

Item 34. A Bioanalytical Mixture Comprising:

-   -   a. at least two nanoparticles;    -   b. a protein in at least two phases; and    -   c. a salt or a buffer.

Item 35. The bioanalytical mixture of item 34, wherein the first of theat least two phases of the protein is a soluble phase.

Item 36. The bioanalytical mixture of item 34 or 35, wherein the secondof the at least two phases of the protein is an adherent phase, whereinthe protein is adhered to the surface of each of the at least twonanoparticles.

Item 37. The bioanalytical mixture according to any one of the precedingitems 34 to 36, wherein the third of the at least two phases of theprotein is an aggregated phase, wherein the protein is self-associatedto form an aggregate.

Item 38. The bioanalytical mixture according to any one of the precedingitems 34 to 37, wherein one or more of the aggregated protein is alsoadhered to the surface of a nanoparticle.

Item 39. The bioanalytical mixture according to any one of the precedingitems 34 to 38, wherein each of the at least two nanoparticles comprisesgold.

Item 40. The bioanalytical mixture according to any one of the precedingitems 34 to 39, wherein each of the at least two nanoparticles comprisesa diameter of about 20 nm to about 100 nm.

Item 41. The bioanalytical mixture according to any one of the precedingitems 34 to 40, wherein the diameter is about 20 nm.

Item 42. The bioanalytical mixture according to any one of the precedingitems 34 to 41, wherein each of the at least two nanoparticles issaturated with the protein.

Item 43. The bioanalytical mixture according to any one of the precedingitems 34 to 42, wherein the nanoparticles are present at a density ofabout 6×10¹¹ to about 7×10¹¹ nanoparticles per milliliter of mixture.

Item 44. The bioanalytical mixture according to any one of the precedingitems 34 to 43, wherein the protein is present at a concentration ofabout 2 μg/mL to about 512 μg/mL.

Item 45. The bioanalytical mixture according to any one of the precedingitems 34 to 44, wherein the protein is an antigen-binding protein.

Item 46. The bioanalytical mixture according to any one of the precedingitems 34 to 45, wherein the antigen-binding protein is selected from thegroup consisting of antibody, antibody fragment, aptamer andreceptor-Fc-fusion protein.

Item 47. The bioanalytical mixture according to any one of the precedingitems 34 to 46, wherein the antigen-binding protein is an antibody.

Item 48. The bioanalytical mixture according to any one of the precedingitems 34 to 47, wherein the antibody is a human monoclonal antibody.

Item 49. The bioanalytical mixture according to any one of the precedingitems 34 to 48, wherein the salt is present at a concentration of about2 mM to about 300 mM.

Item 50. The bioanalytical mixture according to any one of the precedingitems 34 to 49, wherein the salt is present at a concentration of about2 mM, about 20 mM, or about 200 mM.

Item 51. The bioanalytical mixture according to any one of the precedingitems 34 to 50, wherein the salt comprises NaCl.

Examples

The following examples are provided to further illustrate the methods ofthe present invention. These examples are illustrative only and are notintended to limit the scope of the invention in any way.

Example 1: Materials

The monoclonal antibodies (mAbs) were produced by EESYR® cells andpurified by protein A chromatography and a polishing step of anionexchange or hydrophobic interaction chromatography. Buffer componentswere obtained from Sigma-Aldrich, VWR, or JT-Baker and were the highestgrade available. Illustra NAP Columns (Cat. #17-0853-02) and a XK26/100Superdex 200 pg column (Cat. #90-1002-73) were purchased from GEHealthcare Life Sciences. Amicon Centrifugal Filter Units (Cat.#UFC905024) were purchased from EMD Millipore. Slide-A-LyzerTM G2Dialysis Cassettes (Cat. #87732) were purchased from ThermoFisherScientific. 20 nm gold nanoparticles (Cat. #HD.GC20) was purchased fromBBI solutions. Thermo ScientificTM NuncTM MicrowellTM 96-wellmicroplates (Cat. #12-565-66) were used as the reaction container toacquire absorbance spectra.

Example 2: High Concentration Static Light Scattering (HC-SLS)

Concentrated (100 g/L) monoclonal antibodies (mAb1, mAb2, mAb3, mAb4,mAb5 and mAb6) were each purified on an Äkta avant (GE Healthcare LifeSciences) through a XK26/100 Superdex 200 pg column in 10 mM MES pH 6.050 mM sodium chloride. The monomer mAb fraction was collected andconcentrated using an Easy-Load MAsterFlex L/S (Cole Parmer) pump intandem with a VivaFlow 200 30,000 MWCO HY membrane. 150 mL of eachconcentrated antibody was split in to 3 fractions and loaded in to a10,000 MWCO dialysis cassette and exchanged against 2 L of 10 mM MES pH6.0, 250 mM sodium chloride; 10 mM MES pH 6.0, 50 mM sodium chloride;and 10 mM MES pH 6.0, 10 mM sodium chloride solutions. The solutionswere concentrated, using a centrifugal filter unit with a 50,000 MWCO,to a final volume of approximately 15 mL in their respective buffers.The concentrations were measured using a SoloVPE (C Technologies, Inc.).Antibody could not be concentrated beyond nominally 60 g/L in 10 mM MESpH 6.0, 10 mM sodium chloride. The two other salt concentrations (250 mMand 50 mM sodium chloride) were adjusted to nominally 80 g/L. An aliquotfrom the three conditions were taken and diluted to 10 g/L. A sample of80 g/L, 10 g/L and buffer were affixed to a CG-MALS device (WyattTechnology) and the light scattering signal was measured as a functionof mAb concentration.

Example 3: Standard Self-Interaction Nanoparticle Spectroscopy (SINS)

A sub-aliquot of each antibody (mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6)under a different salt condition was added to a separate 15 mL falcontube. 5 mL of one optical density (1 O.D.) of 20 nm gold nanoparticlesolution was added to the solution so that the resultant final proteinconcentration was 50 μg/mL. The absorbance spectra and λ_(max) wererecorded on a SPECTRAmax 340PC (Molecular Devices) after waiting 30minutes.

Concentration-dependent self-interaction nanoparticle spectroscopy(CD-SINS). 100 mM buffer solutions were prepared at the appropriate pH.Illustra NAP columns were conditioned with 2.4 mL of the 100 mM buffersolution (e.g. MES or sodium phosphate depending on the target pH).Concentrated (50-75 g/L) mAb stock solutions were buffer exchanged usingthe conditioned desalting column. The resultant concentration wasmeasured using a SoloVPE. Each antibody was subsequently diluted to 5.12mg/mL in 100 mM buffer. The mAb was then added to column 1 of themicrowellplate and serially diluted in to 100 mM buffer down to 0.04mg/mL. 80 μL of gold nanoparticles was added to columns 2, 3 and 4 ofthe 96 microwellplate. 40 μL of the serially diluted mAb was added toeach column, using a multichannel pipette, containing the goldnanoparticles maintaining the serial dilution from column 1. 280 mL of357 mM, 71 mM and 14 mM sodium chloride salt stock was subsequentlyadded, using a multichannel pipette, to columns 2, 3 and 4 respectively.The resulting solution was allowed to equilibrate for 30 minutes beforethe absorbance spectra was recorded on a SPECTRAmax 340PC. The maximumabsorbance intensity was normalized relative to the absorbance at 450nm. This ratio is plotted as a function of the final concentration ofthe antibody.

Example 4: Dynamic Colloidal Stability of Human Serum Albumin (HSA)

20 nm gold nanoparticles at about 6.3×10¹¹ particles per milliliter werecombined with various concentrations of human serum albumin (HAS) in 10mM MES buffer at 2 mM NaCl, 20 mM NaCl, or 200 mM NaCl, pH 6. Individualsamples containing 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50μg/mL, 100 μg/mL, 200 μg/mL, and 400 μg/mL were subjected to absorbancespectroscopy. Those spectrograms were plotted (FIG. 4, depicts thespectra for the 200 mM NaCl samples) and the ratio of absorbanceintensity at the maximum absorbance (λ_(max)) to the initial absorbanceat 450 nm was calculated for each protein concentration and plotted(FIG. 5).

HSA shows an absorbance intensity ratio greater than 1.7 (A_(peak)/A₄₅₀)for all ionic strengths and at the higher protein concentrations,indicating a favorable dynamic colloidal interaction profile. Staticlight scattering experiments show positive virials (A₂) in overallagreement with the CD-SINS profile (Table 2).

TABLE 2 NaCl mM SLS (A₂) 2 1.17E−4 20 9.68E−5 120 7.85E−5

Example 5: Monoclonal Antibody No. 1 (mAb1)

20 nm gold nanoparticles at about 6.3×10¹¹ particles per milliliter werecombined with various concentrations of human mAb1 in 10 mM MES bufferat 2 mM NaCl, 20 mM NaCl, or 200 mM NaCl, pH 6. Individual samplescontaining 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100μg/mL, 200 μg/mL, and 400 μg/mL were subjected to absorbancespectroscopy. Those spectrograms were plotted and the ratio ofabsorbance intensity at the maximum absorbance (λ_(max)) to the initialabsorbance at 450 nm was calculated for each protein concentration andplotted (FIG. 6).

mAb1 shows an absorbance intensity ratio greater than 1.7(A_(peak)/A₄₅₀) for all ionic strengths and at the higher proteinconcentrations, indicating a favorable dynamic colloidal interactionprofile. High-concentration static light scattering experiments showpositive virials (A₂) in overall agreement with the CD-SINS profile(Table 3, FIG. 7).

TABLE 3 NaCl mM SLS (A₂) 2 1.72E−4 20 6.70E−5 200 1.34E−5

Example 6: Monoclonal Antibody No. 6 (mAb6)

20 nm gold nanoparticles at about 6.3×10¹¹ particles per milliliter werecombined with various concentrations of human mAb6 in 10 mM MES bufferat 2 mM NaCl, 20 mM NaCl, or 200 mM NaCl, pH 6. Individual samplescontaining 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100μg/mL, 200 μg/mL, and 400 μg/mL were subjected to absorbancespectroscopy. Those spectrograms were plotted and the ratio ofabsorbance intensity at the maximum absorbance (μ_(max)) to the initialabsorbance at 450 nm was calculated for each protein concentration andplotted (FIG. 8).

mAb6 shows an absorbance intensity ratio greater than 1.7(A_(peak)/A₄₅₀) at the lower ionic strengths and at the higher proteinconcentrations, indicating a favorable dynamic colloidal interactionprofile under those conditions. mAb6 exhibits an increase in attractiveinteractions with increasing ionic strength. High-concentration staticlight scattering experiments show variable virials (A₂) in overallagreement with the CD-SINS profile (FIG. 9).

Example 7: Monoclonal Antibody No. 2 (mAb2)

20 nm gold nanoparticles at about 6.3×10¹¹ particles per milliliter werecombined with various concentrations of human mAb2 in 10 mM MES bufferat 2 mM NaCl, 20 mM NaCl, or 200 mM NaCl, pH 6. Individual samplescontaining 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100μg/mL, 200 μg/mL, and 400 μg/mL were subjected to absorbancespectroscopy. Those spectrograms were plotted and the ratio ofabsorbance intensity at the maximum absorbance (λ_(max)) to the initialabsorbance at 450 nm was calculated for each protein concentration andplotted (FIG. 2).

mAb2 shows profound changes in net protein interactions under varyingconditions. At low ionic strength, mAb2 exhibits an increase inattractive interactions. The molecule becomes overall repulsive as theionic strength increases. At 200 mM NaCl, the antibody has favorablecolloidal stability. High-concentration static light scatteringexperiments show variable virials (A₂) in overall agreement with theconventional SINS wavelengths of maximum absorbance (Table 1).

Example 8: Monoclonal Antibody No. 3 (mAb3)

20 nm gold nanoparticles at about 6.3×10¹¹ particles per milliliter werecombined with various concentrations of human mAb3 in 10 mM MES bufferat 2 mM NaCl, 20 mM NaCl, or 200 mM NaCl, pH 6. Individual samplescontaining 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100μg/mL, 200 μg/mL, and 400 μg/mL were subjected to absorbancespectroscopy. Those spectrograms were plotted and the ratio ofabsorbance intensity at the maximum absorbance (λ_(max)) to the initialabsorbance at 450 nm was calculated for each protein concentration andplotted (FIG. 10).

mAb3 is an example of a colloidally unstable protein that portendsproblematic high concentration formulation and manufacturing. At allionic strengths and protein concentrations, mAb3 exhibits attractiveinteractions. High-concentration static light scattering experimentsshow negative virials (A₂) in overall agreement with the conventionalSINS wavelengths of maximum absorbance (Table 1).

Example 9: Monoclonal Antibody No. 4 (mAb4)

20 nm gold nanoparticles at about 6.3×10¹¹ particles per milliliter werecombined with various concentrations of human mAb4 in 10 mM MES bufferat 2 mM NaCl, 20 mM NaCl, or 200 mM NaCl, pH 6. Individual samplescontaining 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100μg/mL, 200 μg/mL, and 400 μg/mL were subjected to absorbancespectroscopy. Those spectrograms were plotted and the ratio ofabsorbance intensity at the maximum absorbance (λ_(max)) to the initialabsorbance at 450 nm was calculated for each protein concentration andplotted (FIG. 11).

mAb4 is an example of a colloidally stable protein at lower ionicstrengths, but showing less solubility at higher protein concentrationin low ionic strength. Static light scattering experiments, which depictmixed virials (negative at lower ionic strength, positive at high ionicstrength), show discrepancies with the CD-SINS-generated data, whichshows a repulsive profile for mAb4 (Table 1).

Example 10: Monoclonal Antibody No. 5 (mAb5)

20 nm gold nanoparticles at about 6.3×10¹¹ particles per milliliter werecombined with various concentrations of human mAb5 in 10 mM MES bufferat 2 mM NaCl, 20 mM NaCl, or 200 mM NaCl, pH 6. Individual samplescontaining 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100μg/mL, 200 μg/mL, and 400 μg/mL were subjected to absorbancespectroscopy. Those spectrograms were plotted and the ratio ofabsorbance intensity at the maximum absorbance (λ_(max)) to the initialabsorbance at 450 nm was calculated for each protein concentration andplotted (FIG. 12).

mAb5 is an example of a colloidally mixed protein that is attractive inlow ionic strength and exhibits improvement in high proteinconcentration behavior with increasing ionic strength. Static lightscattering experiments, which depict only negative virials, showdiscrepancies with the CD-SINS-generated data, which shows a mixedprofile for mAb4 (Table 1).

Example 11: Excipient Screening and Selection

A high-viscosity formulated antibody having an unfavorable dynamiccolloidal interaction profile under some formulation conditions (mAb7)was used to screen additional excipients for their ability to improvethe antibody's CG-SINS profile. The CG-SINS profile of monoclonalantibody 7 (mAb7) formulated in various solutions containing differentbuffers and ionic strengths (FIG. 13), and sugar and/or amino acidexcipients (FIG. 14) remained below the absorbance_(max)/absorbance₀threshold value favorable for dispensing at high concentration (e.g.,below 1.7). The viscosity of mAb7 formulated at about 100 mg/mL wasobserved at ˜100 cP.

The CD-SINS assay was used to screen the colloidal stabilizing effect ofvarious substituted benzoate compounds on mAb7. Substituted benzoicacids include m-hydroxybenzoic acid, ρ-hydroxybenzoic acid,m-methylbenzoic acid, ρ-methylbenzoic acid, m-ethylbenzoic acid,ρ-ethylbenzoic acid, m-aminobenzoic acid, ρ-aminobenzoic acid, asulfonic acid, and an ammonium benzoate. FIG. 15 depicts the CD-SINSabsorbance ratio scatter plot of mAb7 formulated with each of thesubstituted benzoic acids. When ρ-aminobenzoic acid (PABA, open circlesin FIG. 15) was combined with the formulated mAb7, the mAb7 exhibitedfavorable colloidal stability as demonstrated by absorbance ratios overthe threshold value favorable for dispersing at high concentration,i.e., >1.6 or 1.7 as depicted in FIG. 15.

PABA was combined in a dose dependent manner with mAb7 and subjected toCD-SINS analysis. The inclusion of >12 mM PABA (e.g., 18 Mm, 24 mM, 30mM, and 36 mM PABA) with mAb7 produced a favorable (i.e., repulsive)colloidal interaction profile (FIG. 16). The effect of PABA on thecolloidal interaction profile of mAb7 appeared to saturate atapproximately 20 mM PABA.

20 mM PABA was combined with 5 g/L, 10 g/L, 30 g/L, 50 g/L, 70 g/L and80 g/L of mAb7 and subjected to viscometric analysis using the cone andplate method using a torsional rheometer (Pathak et al., Biophys. J.104(4): 913-923, 2013 Feb 19). The results are depicted in FIG. 17.Extrapolation of those results to 100 g/L mAb7 using theKrieger-Dougherty algorithm (see, e.g., Lukham and Ukeje, J. ColloidInterface Sci., 220(2): 347-356, 1999 Dec 15) showed an approximatelythree-fold reduction in steady shear viscosity in those mAb7formulations containing 20 mM PABA.

1-45. (canceled)
 46. A method of making a low viscosity pharmaceuticalformulation containing a protein, the method comprising: a. determiningthe potential of the protein to self-associate; b. combining aviscosity-reducing excipient with the protein; c. determining thepotential of the protein to self-associate in the presence of theviscosity-reducing excipient; and d. formulating the protein with theviscosity-reducing excipient at a level that reduces the viscosity ofthe protein solution by at least 50% than without the viscosity-reducingexcipient.
 47. The method of claim 46, wherein the viscosity-reducingexcipient is para-aminobenzoic acid (PABA).
 48. The method of claim 46,wherein determining the potential of the protein to self-associateincludes determining the potential of the protein to self-associate whenit is at a high concentration.
 49. The method of claim 48, whereindetermining the potential of the protein to self-associate when it is ata high concentration comprises: a. combining the protein at a lowconcentration, a nanoparticle, and a buffered salt to form a sample; b.exciting the sample with light; c. measuring the light transmittedthrough the sample at multiple wavelengths ranging from 450 nm to about750 nm; d. calculating the absorbance intensity ratio of the sample,wherein the absorbance intensity ratio is the ratio of absorbanceintensity at the maximum absorbance (λmax) to the initial absorbance at450 nm; e. measuring the absorbance intensity ratio, and f. determiningthe protein retains 90% or more of its native structure at a highconcentration when the absorbance intensity ratio exceeds 1.7.
 50. Themethod of claim 49, wherein: (i) the protein is an antigen-bindingprotein; (ii) the protein is in the sample at the low concentration ofabout 2 μg/mL to about 512m/mL; (iii) the nanoparticle is a goldnanoparticle, wherein the gold nanoparticle has a diameter of about 20nm to about 100 nm; (iv) the sample comprises about 5×10¹¹ to about8×10¹¹ nanoparticles per mL; and/or (v) the salt is present in thesample at a concentration of about 2 mM to about 250 mM.
 51. The methodof claim 49 further comprising repeating steps (a)-(f) using a differentconcentration of protein in the sample.
 52. The method of claim 49further comprising repeating steps (a)-(f) using a differentconcentration of salt in the sample.
 53. The method of claim 49 furthercomprising repeating steps (a)-(f) using a different pH of the sample.54. The method of claim 49 further comprising: (g) combining the proteinat a high concentration with an excipient to form a formulated drugsubstance or drug product.
 55. The method of claim 54, wherein theconcentration of the protein is about 50 mg/mL to about 500 mg/mL. 56.The method of claim 54, wherein the excipient is selected from the groupconsisting of a tonicifier, a buffer, a surfactant, stabilizer, and acombination thereof.
 57. The method of claim 49, wherein the protein ispresent in the sample in a concentration in excess of a minimumconcentration necessary to completely cover the nanoparticles.
 58. Themethod of claim 49, wherein the protein is a receptor-Fc-fusion protein.59. The method of claim 49, wherein the protein is an antibody orantibody fragment.
 60. The method of claim 51, wherein the differentprotein concentrations include two or more protein concentrations fromabout 64m/mL to about 512m/mL.
 61. The method of claim 48, wherein theprotein is at a high concentration when it is present in a formulationat a concentration between about 50 mg/mL to about 500 mg/mL.
 62. Themethod of claim 48, wherein the protein is at a high concentration whenit is present in a formulation at a concentration between about 50 mg/mLto about 250 mg/mL.
 63. The method of claim 46, wherein the samplecomprises about 6×10¹¹ to about 6.5×10¹¹ nanoparticles per mL.
 64. Themethod of claim 49, wherein: (i) the protein is in the sample at aconcentration of about 2 μm/mL to about 512 μg/mL; and (ii) the proteinis at a concentration when it is present in a formulation at aconcentration between about 50 mg/mL to about 500 mg/mL.
 65. The methodof claim 49, wherein: (i) the protein is an antigen-binding protein, areceptor-Fc-fusion protein, an antibody, or antibody fragment; (ii) theprotein is in the sample at a concentration of about 2 μm/mL to about512 μg/mL; (iii) the nanoparticle is a gold nanoparticle that has adiameter of about 20 nm to about 100 nm; (iv) the sample comprises about5×10¹¹ to about 8×10¹¹ nanoparticles per mL; (v) the salt is present inthe sample at a concentration of about 2 mM to about 250 mM; and (vi)the protein is at a high concentration when it is present in aformulation at a concentration between about 50 mg/mL to about 500mg/mL.